It is unclear, though, how John might have had access to the works of the Rhineland Mystics. These works were only translated into Latin in the second half of the sixteenth century. This means that copies would hardly have been easily available for John. In addition, if it is acknowledged that an influence on John was Francisco de Osuna (who clearly cannot have read the Rhineland Mystics since they were not known in Spain in the 1520s), then another Problem is raised.

He was born Juan de Yepes y Álvarez into a converso family (descendents of Jewish converts to Christianity) in Fontiveros, near Ávila, a town of around 2,000 people. His father, Gonzalo, was an accountant to richer relatives who were silk merchants. However, when in 1529 he married John's mother, Catalina, who was an orphan of a lower class, Gonzalo was rejected by his family and forced to work with his wife as a weaver. John's father died in 1545, while John was still only around three years old. Two years later, John's older brother Luis died, probably as a result of insufficient nourishment caused by the penury to which John's family had been reduced. After this, John's mother Catalina took John and his surviving brother Francisco, and moved first in 1548 to Arévalo, and then in 1551 to Medina del Campo, where she was able to find work weaving.

In Medina, John entered a school for around 160 poor children, usually orphans, receiving a basic education, mainly in Christian doctrine, as well as some food, clothing and lodging. While studying there, he was chosen to serve as acolyte at a nearby monastery of Augustinian nuns. Growing up, John worked at a hospital and studied the humanities at a Jesuit school from 1559 to 1563; the Society of Jesus was a new organization at the time, having been founded only a few years earlier by the Spaniard St. Ignatius of Loyola. In 1563 he entered the Carmelite Order, adopting the name John of St. Matthias.

In order to gain a better understanding of the intellectual influences to which John was exposed in his formative years (and so to isolate what shaped his unusual theology), many scholars have tried to reconstruct John's likely course of studies while he was at Salamanca between 1563 and 1567, living at the Carmelite College of San Andrès and studying at the University of Salamanca. It has been widely acknowledged in the 20th century to be most likely that John would have received teaching both from the College of San Andres and from Salamanca University.

The following year (1564) he professed his religious vows as a Carmelite and travelled to Salamanca, where he studied theology and philosophy at the prestigious University there (at the time one of the four biggest in Europe, alongside Paris, Oxford and Bologna) and at the Colegio de San Andrés. Some modern Writers claim that this stay would influence all his later writings, as Fray Luis de León taught biblical studies (Exegesis, Hebrew and Aramaic) at the University: León was one of the foremost experts in Biblical Studies then and had written an important and controversial translation of the Song of Songs into Spanish. (Translation of the Bible into the vernacular was not allowed then in Spain, because of the possibility of mistranslation from Latin to Spanish which could create confusion.)

John was ordained a priest in 1567, and then indicated his intent to join the strict Carthusian Order, which appealed to him because of its encouragement of solitary and silent contemplation. A journey from Salamanca to Medina del Campo, probably in September 1567, changed this. In Medina he met the charismatic Carmelite nun Teresa of Jesus. She was in Medina to found the second of her convents for women. She immediately talked to him about her reformation projects for the Order: she was seeking to restore the purity of the Carmelite Order by restarting observance of its "Primitive Rule" of 1209, observance of which had been relaxed by Pope Eugene IV in 1432.

Teresa asked John to delay his entry into the Carthusians and to follow her. Having spent a final year studying in Salamanca, in August 1568 John traveled with Teresa from Medina to Valladolid, where Teresa intended to found another monastery of nuns. Having spent some time with Teresa in Valladolid, learning more about this new form of Carmelite life, in October 1568, accompanied by Friar Antonio de Jesús de Heredia, John left Valladolid to found a new monastery for friars, the first for men following Teresa's principles. They were given the use of a derelict house at Duruelo (midway between Ávila and Salamanca), which had been donated to Teresa. On 28 November 1568, the monastery was established, and on that same day John changed his name to "John of the Cross".

Soon after, in June 1570, the friars found the house at Duruelo too small, and so moved to the nearby town of Mancera de Abajo. After moving on from this community, John set up a new community at Pastrana (October 1570), and a community at Alcalá de Henares, which was to be a house of studies for the academic training of the friars. In 1572 he arrived in Ávila, at the invitation of Teresa, who had been appointed prioress of the Monastery of the Incarnation there in 1571. John became the spiritual Director and confessor for Teresa and the other 130 nuns there, as well as for a wide range of laypeople in the city. In 1574, John accompanied Teresa in the foundation of a new monastery in Segovia, returning to Avila after staying there a week. Beyond this, though, John seems to have remained in Ávila between 1572 and 1577.

However, the belief that John was taught at both the Carmelite College of San Andrès and at the University of Salamanca has been criticised. He argues, firstly, that it is unclear whether there were in fact lectures in arts and theology at the College of San Andrès before 1571, that a reconstruction of the typical timetable of teaching at the University of Salamanca shows that there would have been little time for extra teaching at the College of the San Andrès, and that what therefore happened at San Andrès (if indeed it happened at all) is therefore likely to have taken the form of rehearsals, preparation of classes, and not in an official form — making the idea that John received systematic exposure to Baconthorpe less likely. More controversially, Bezares calls into question whether John even studied theology at the University of Salamanca. The philosophy courses John probably took in logic, natural and moral philosophy, can be reconstructed, but Bezares argues that John in fact abandoned his studies at Salamanca in 1568 to join Teresa, rather than having graduated, meaning that he did not study theology in Salamanca.

At some point between 1574 and 1577, while praying in the Monastery of the Incarnation in Ávila in a loft overlooking the sanctuary, John had a vision of the crucified Christ, which led him to create his famous drawing of Christ "from above". In 1641, this drawing was placed in a small monstrance and kept in Ávila. This drawing inspired the Artist Salvador Dalí's 1951 work Christ of Saint John of the Cross.

Others, though, have questioned the evidence for precisely how John might have been influenced by Boscan and Garcilaso. Dámaso Alonso argued that John must have read the newly published 1575 edition of the poets in Ávila, shortly before his imprisonment in Toledo, and that this must have been the key influence which rekindled in John memories of his own reading of Garcilaso as a young student of the Jesuits in Medina del Campo. However, Peter Thompson disputes this, arguing it is not definite John would have been familiar with Garcilaso from an early age, and even so the influence has been overemphasised by other commentators.

In Andalusia to the south, however, where the Visitor was Francisco Vargas, tensions rose due to his clear preference for the Discalced friars. Vargas asked them to make foundations in various cities, in explicit contradiction of orders from the Carmelite Prior General against their expansion in Andalusia. As a result, a General Chapter of the Carmelite Order was convened at Piacenza in Italy in May 1576, out of concern that events in Spain were getting out of hand, which concluded by ordering the total suppression of the Discalced houses.

On the night of 2 December 1577, a group of Carmelites opposed to reform broke into John's dwelling in Ávila and took him prisoner. John had received an order from some of his superiors, opposed to reform, ordering him to leave Ávila and return to his original house, but John had refused on the basis that his reform work had been approved by the Spanish nuncio, a higher authority than these superiors. The Carmelites therefore took John captive. John was taken from Ávila to the Carmelite monastery in Toledo, at that time the Order's most important monastery in Castile, where perhaps 40 friars lived. John was brought before a court of friars, accused of disobeying the ordinances of Piacenza. Despite John's argument that he had not disobeyed the ordinances, he received a punishment of imprisonment. He was jailed in the monastery, where he was kept under a brutal regimen that included public lashing before the community at least weekly, and severe isolation in a tiny stifling cell measuring ten feet by six feet, barely large enough for his body. Except when rarely permitted an oil lamp, he had to stand on a bench to read his breviary by the light through the hole into the adjoining room. He had no change of clothing and a penitential diet of water, bread and scraps of salt fish. During this imprisonment, he composed a great part of his most famous poem Spiritual Canticle, as well as a few shorter poems. The paper was passed to him by the friar who guarded his cell. He managed to escape nine months later, on 15 August 1578, through a small window in a room adjoining his cell. (He had managed to pry the cell door off its hinges earlier that day.)

The Dark Night (from which the spiritual term takes its name) narrates the journey of the soul from its bodily home to union with God. It happens during the night, which represents the hardships and difficulties met in detachment from the world and reaching the light of the union with the Creator. There are several steps in this night, which are related in successive stanzas. The main idea of the poem can be seen as the painful experience that people endure as they seek to grow in spiritual maturity and union with God. The poem of this title was likely written in 1578 or 1579. In 1584-5, John wrote a commentary on the first two stanzas and first line of the third stanza of the poem.

In 1579 he moved to Baeza, a town of around 50,000 people, to serve as rector of a new college, the Colegio de San Basilio, to support the studies of Discalced friars in Andalusia. This opened on 13 June 1579. He remained in post there until 1582, spending much of his time as a spiritual Director for the friars and townspeople.

The Ascent of Mount Carmel is a more systematic study of the ascetical endeavour of a soul looking for perfect union, God and the mystical events happening along the way. Although it begins as a commentary on the poem The Dark Night, it rapidly drops this format, having commented on the first two stanzas of the poem, and becomes a treatise. It was composed sometime between 1581 and 1585.

A four-stanza work, Living Flame of Love, describes a greater intimacy, as the soul responds to God's love. It was written in a first redaction at Granada between 1585-6, apparently in two weeks, and in a mostly identical second redaction at La Peñuela in 1591.

In June 1588, he was elected third Councillor to the Vicar General for the Discalced Carmelites, Father Nicolas Doria. To fulfill this role, he had to return to Segovia in Castile, where in this capacity he was also prior of the monastery. After disagreeing in 1590–1 with some of Doria's remodeling of the leadership of the Discalced Carmelite Order, though, John was removed from his post in Segovia, and sent by Doria in June 1591 to an isolated monastery in Andalusia called La Peñuela. There he fell ill, and traveled to the monastery at Úbeda for treatment. His condition worsened, however, and he died there on 14 December 1591, of erysipelas.

The morning after John’s death huge numbers of the townspeople of Úbeda entered the monastery to view his body; in the crush, many were able to take home parts of his habit. He was initially buried at Úbeda, but, at the request of the monastery in Segovia, his body was secretly moved there in 1593. The people of Úbeda, however, unhappy at this change, sent a representative to petition the pope to move the body back to its original resting place. Pope Clement VIII, impressed by the petition, issued a Brief on 15 October 1596 ordering the return of the body to Ubeda. Eventually, in a compromise, the superiors of the Discalced Carmelites decided that the monastery at Úbeda would receive one leg and one arm of the corpse from Segovia (the monastery at Úbeda had already kept one leg in 1593, and the other arm had been removed as the corpse passed through Madrid in 1593, to form a relic there). A hand and a leg remain visible in a reliquary at the Oratory of San Juan de la Cruz in Úbeda, a monastery built in 1627 though connected to the original Discalced monastery in the town founded in 1587.

Proceedings to beatify John began with the gathering of information on his life between 1614 and 1616, although he was only beatified in 1675 by Pope Clement X, and was canonized by Benedict XIII in 1726. When his feast day was added to the General Roman Calendar in 1738, it was assigned to 24 November, since his date of death was impeded by the then-existing octave of the Feast of the Immaculate Conception. This obstacle was removed in 1955 and in 1969 Pope Paul VI moved it to the dies natalis (birthday to heaven) of the saint, 14 December. The Church of England commemorates him as a "Teacher of the Faith" on the same date. In 1926, he was declared a Doctor of the Church by Pope Pius XI after the definitive consultation of Reginald Garrigou-Lagrange O.P., professor of philosophy and theology at the Pontifical University of Saint Thomas Aquinas, Angelicum in Rome.

His writings were first published in 1618 by Diego de Salablanca. The numerical divisions in the work, still used by modern editions of the text, were introduced by Salablanca (they were not in John's original writings) in order to help make the work more manageable for the reader. This edition does not contain the Spiritual Canticle however, and also omits or adapts certain passages, perhaps for fear of falling foul of the Inquisition.

The first French edition was published in Paris in 1622, and the first Castilian edition in 1627 in Brussels.

Another claim frequently made about John's time in Salamanca, by scholars trying to explain the origins of John's mystical thought, is that it was here he was exposed in detail to mystical thought. In the first biography of John, published in 1628, it is claimed, on the basis of information from John's fellow students, that he in 1567 made a special study of mystical Writers, in particular of Pseudo-Dionysius and Saint Gregory the Great. Much weight has been put on this evidence by later Writers. However, others have doubted the veracity of this anecdote, even though they do not dispute that John may have studied mystical theology in this period.

The Spiritual Canticle was first included in the 1630 edition, produced by Fray Jeronimo de San José, at Madrid. This edition was largely followed by later editors, although editions in the seventeenth and eighteenth centuries gradually included a few more poems and letters.

The head and torso were retained by the monastery at Segovia. There, they were venerated until 1647, when on orders from Rome designed to prevent the veneration of remains without official approval, the remains were buried in the ground. In the 1930s they were disinterred, and now sit in a side chapel in a marble case above a special altar built in that decade.

John of the Cross is known for his writings. Both his poetry and his studies on the growth of the soul are considered the summit of mystical Spanish literature and one of the peaks of all Spanish literature. He was canonized as a saint in 1726 by Pope Benedict XIII. He is one of the thirty-six Doctors of the Church.

However, a strong argument can also be made for contemporary Spanish literary influences on John. This case was first made in detail by Dámaso Alonso, who believed that as well as drawing from scripture, John was transforming non-religious, profane themes, derived from popular songs (romanceros) into religious poetry; Alonso argued that John was particularly influenced by the works of the Spanish Renaissance poets Garcilaso de la Vega and Boscán. Certainly, John does appear to have used Garcilaso's verse forms in his own poetry, in particular in the Spiritual Canticle, Dark Night, and Living Flame of Love. Alongside the various biblical images noted above, for Example, exist in John's poems many new Renaissance symbols of pastoral love, prominent in the poetry of Garcilaso and Boscán, such as sirens, nightingales, nymphs, doves and Shepherds. Moreover, in the Prologue to the Living Flame, John states that "the composition of these lyric lines is like those that in Boscán are given a religious meaning". Kavanaugh (1991) also points out that these lines are in fact not by Boscán, but by Garcilaso, although the confusion was Common at the time, as the works of the two poets had been published together.

John was clearly influenced by the Bible. Scriptural images are Common in both his poems and prose—in total, there are 1,583 explicit and 115 implicit quotations from the Bible in his works. The influence of the Song of Songs on the Spiritual Canticle has often been noted, both in terms of the structure of the poem, with its dialogue between two lovers, the account of their difficulties in meeting each other and the "offstage chorus" that comments on this action, and also in terms of the imagery of pomegranates, wine cellar, turtle dove and lilies, for Example, which echoes that of the Song of Songs.

A controversial theory of the origins of John’s mystical imagery is that he was influenced by Islamic sources. This was first proposed in detail by Miguel Asín Palacios and has been most recently put forward by the Puerto Rican scholar Luce Lopez Baralt. Arguing that John was influenced by Islamic sources on the peninsula, she traces Islamic antecedents of the images of the "dark night", the "solitary bird" of the Spiritual Canticle, wine and mystical intoxication (the Spiritual Canticle), lamps of fire (the Living Flame) and so on.