Born on 1 September 1896, the day after Janmastami ,one of the most important Vaishnava holidays, in a humble house in the Tollygunge suburb of Calcutta in a Bengali Suvarna Banik family, he was named Abhay Charan, "one who is fearless, having taken shelter at Lord Krishna's feet." Since he was born on the day of Nandotsava ("the celebration of Nanda," Krishna's father, a traditional festival in honour of Krishna's birth) he was also called Nandulal. His parents, Sriman Gour Mohan De and Srimati Rajani De, were devout Vaishnavas (devotees of Vishnu). In accordance with Bengali tradition, his mother had gone to the home of her parents for the delivery, and only a few days later Abhay returned with parents to his home at 6 Sitakanta Banerjee Lane Kolkata 700005

He received a European-led education in the Scottish Church College, which was well reputed among Bengalis; many Vaishnava families sent their sons there. The professors, most of whom were Europeans, were known as sober, moral men, and it is believed that the students received a good education. The college was located in north Calcutta, near the De's family home on Harrison Road. During his years in the college, Abhay Charan De (অভয় চরণ দে) was a member of the English Society as well as that of the Sanskrit Society, and it has been suggested that his education provided him a foundation for his Future leadership. He graduated in 1920 with majors in English, philosophy and economics. He rejected his diploma in response to Gandhi's independence movement.

In 1922, when he first met his spiritual master, Bhaktisiddhanta Sarasvati Thakura, he was requested to spread the message of Chaitanya Mahaprabhu in the English language. In 1933 he became a formally initiated disciple of Bhaktisiddhanta. In 1944, (from his front room at Sita Kanta Banerjee, Calcutta), he started the publication called Back to Godhead, for which he acted as Writer, designer, publisher, Editor, copy Editor and distributor. He personally designed the logo, an effulgent figure of Chaitanya Mahaprabhu in the upper left corner, with the motto: "Godhead is Light, Nescience is darkness" greeting the readers. In his first magazine he wrote:

In 1947, the Gaudiya Vaishnava Society recognised his scholarship with the title Bhaktivedanta, (bhakti-vedānta) meaning "one who has realised that devotional Service to the Supreme Lord is the end of all knowledge" (with the words Bhakti, indicating devotion and Vedanta indicating conclusive knowledge).

From 1950 onwards, he lived at the medieval Radha-Damodar mandir in the holy town of Vrindavan, where he began his commentary and translation work of the Sanskrit work Bhagavata Purana. Of all notable Vrindavana's temples, the Radha-Damodara mandir had at the time the largest collection of various copies of the original writings of the Six Gosvamis and their followers – more than two thousand separate manuscripts, many of them three hundred, some even four hundred years old. His guru, Bhaktisiddhanta Sarasvati Thakura, had always encouraged him to print books, and beholding his spiritual master, Abhay felt the words deeply enter his own life – "If you ever get money, print books." referring to the need of literary presentation of the Vaishnava culture.

Beginning his public preaching mission in India, he founded the League of Devotees in Jhansi in 1953.

The Gaudiya Matha at Allahabad, Uttar Pradesh was where he lived, wrote and studied, edited the Gauḍīya Patrikā magazine and where he donated the idol (murti) of Chaitanya Mahaprabhu which stands on the altar beside those of Radha Krishna (named Śrī Śrī Rādhā Vinodavihārījī). During his visit in September 1959 he entered the doors of this matha dressed in white, as Abhoy Babu, but would be leaving dressed in saffron, a Vaishnava renunciate (sannyasi). He took his renunciate vows from his friend and godbrother Bhakti Prajnana Keshava. On becoming a sannyasa he also took the prenominal Swami (स्वामी Svāmi). He single-handedly published the first three volumes covering seventeen chapters of the first book of Bhagavata Purana, filling three volumes of four hundred pages, each with a detailed commentary. The introduction to the first volume was a biographical Sketch of Caitanya Mahāprabhu. He then left India, obtaining free passage on a freighter called the Jaladuta, with the aim and a hope of fulfilling his spiritual master's instruction to spread the message of Caitanya Mahaprabhu around the world. In his possession were a suitcase, an umbrella, a supply of dry cereal, about eight dollars worth of Indian currency, and several boxes of books.

When he sailed to the United States in 1965, his trip was not sponsored by any religious organization, nor was he met upon arrival by a group of loyal followers. As the Indian freighter Jaladuta neared his destination, the magnitude of his intended task weighed on him. On 13 September he wrote in his diary, "Today I have disclosed my mind to my companion, Lord Sri Krishna." On this occasion and on a number of others, he called on Krishna for help in his native Bengali. Examining these compositions, academics regard them as "intimate records of his prayerful preparation for what lay ahead" and a view on "how Bhaktivedanta Swami understood his own identity and mission."

When it was suggested to him at the time of founding the ISKCON in 1966 that a broader term "God Consciousness" would be preferable to "Krishna Consciousness" in the title, he rejected this recommendation, suggesting that the name Krishna includes all other forms and concepts of God.

After a group of devotees and a temple had been established in New York another centre was started in San Francisco in 1967. From there he travelled throughout America with his disciples, popularizing the movement through street chanting (sankirtana), book distribution and public speeches.

It is believed that Bhaktivedanta Swami's most significant contribution are his books. Within the final twelve years of his life Bhaktivedanta Swami translated over sixty volumes of classic Vedic scriptures (such as the Bhagavad Gita, Chaitanya Charitamritaand the Srimad Bhagavatam) into the English language. For their authority, depth, and clarity, his books have won praise from professors at colleges and universities like Harvard, Oxford, Cornell, Columbia, Syracuse, Oberlin, and Edinburgh, and his Bhagavad-Gītā As It Is was published by Macmillan Publishers, in 1968 and unabridged edition in 1972, and is now available in over sixty languages around the world and some other books by Bhaktivedanta Swami are available in over eighty different languages. In February 2014, ISKCON's news agency reported to have reached a milestone of distributing over half a billion books authored by Bhaktivedanta Swami Prabhupada, since 1965.

Following the establishment of temples and centres in the United States and Europe, he returned to India in 1971, holding many public programs which were well attended. From 1971 onwards, the movement became increasingly popular and spread throughout India. He was particularly eager to see the progress at "the impressive temple project in" Mumbai which he and his disciples had fought very hard to establish, with large temples in Mayapur and Vrindavan to follow in the mid-1970s. To promote the vedic education system within the modern Indian education structure, he introduced a chain of Gurukul in various part of India. The Bhaktivedanta Gurukula and International School is one of the most successful schools in the list.

A prominent Gaudiya Vaishnava figure, Shrivatsa Goswami, who as a young man had met Bhaktivedanta Swami in 1972, affirmed the significance of book publishing and distribution in spreading the message of Caitanya in an interview with Steven Gelberg:

Bhaktivedanta Swami died on 14 November 1977 in Vrindavan, India; his burial remains in Krishna Balaram Mandir in Vrindavan India.

A number of memorial samadhis or shrines to Bhaktivedanta Swami were constructed by the members of ISKCON in his remembrance, the largest of which are in Mayapur, Vrindavan and at the larger sized temples in America. Prabhupada's Palace of Gold was designed and constructed by devotees of the New Vrindavan community and dedicated on 2 September 1979. Back in 1972 it was intended to be simply a residence for Bhaktivedanta Swami, but over time the plans evolved into an ornate marble and gold palace which is now visited by thousands of Hindu pilgrims each year, visiting this centrepiece of the community strongly relying upon tourist trade.

In 1996, the Government of India recognized his accomplishments by issuing a commemorative stamp in his honour as a part of Prabhupada Centennial celebrations.

In 1997, a legal dispute opposing ISKCON Inc. / BBTI (Bhaktivedanta Book Trust International) to the sole trustee of Bhaktivedanta Book Trust (at this time, Hansadutta Swami), ISKCON Inc. supported the position that Bhaktivedanta Book Trust was non-existent; in 1995 it was claimed by ISKCON that Bhaktivedanta's work was done as a "Work for hire". The litigation led to amiable arrangement after which books of Bhaktivedanta are being edited in mainly two forms: original work ("Pre 1978") and work which has, since Bhaktivedanta's passing, been further edited, the later being the sole endorsed by ISKCON.

Speaking at the inauguration of ISKCON's cultural centre in New Delhi on 5 April on the occasion of Ramnavmi in 1998, Atal Bihari Vajpayee, then India's prime minister, said: