On 10 November 1509, Pizarro sailed from Spain to the New World with Alonso de Ojeda on an expedition to Urabá. He sailed to Cartagena and joined the fleet of Martín Fernández de Enciso and, in 1513, accompanied Balboa to the Pacific. In 1514, he found a supporter in Pedro Arias Dávila, the Governor of Castilla de Oro and was rewarded for his role in the arrest of Balboa with the positions of mayor and magistrate in Panama City, serving from 1519 to 1523.

In 1513, Pizarro accompanied Vasco Núñez de Balboa in his crossing of the Isthmus of Panama to the Pacific coast. The following year, Pedro Arias Dávila became the newly appointed governor of Castilla de Oro and succeeded Balboa. During the next five years, Pizarro became a close associate of Dávila and the governor assigned him a repartimiento of natives and cattle. When Dávila decided to get rid of Balboa out of distrust, he instructed Pizarro to personally arrest him and bring him to stand trial. Balboa was beheaded in January 1519. For his loyalty to Dávila, Pizarro was rewarded with the positions of mayor (Alcalde) and magistrate of the then recently founded Panama City from 1519 to 1523.

The first attempt to explore western South America was undertaken in 1522 by Pascual de Andagoya. The native South Americans he encountered told him about a gold-rich territory called Virú, which was on a river called Pirú (later corrupted to Perú). These reports were related by the Spanish-Inca mestizo Writer Garcilaso de la Vega in Comentarios Reales de los Incas (1609).

In November 1524, the first of three expeditions left Panama for the conquest of Peru with about 80 men and 40 horses. Juan de Salcedo was the standard bearer, Nicolás de Ribera was the treasurer and Juan Carvallo was the inspector.

On 10 March 1526 Pizarro left Panama with two ships with 160 men and several horses, reaching as far as the Colombian San Juan River. Soon after arriving the party separated, with Pizarro staying to explore the new and often perilous territory off the swampy Colombian coasts, while the expedition's co-commander, Almagro, returned to Panama for reinforcements. Pizarro's Piloto Mayor (main pilot), Bartolomé Ruiz, continued sailing south and, after crossing the equator, found and captured a balsa (raft) under sail, with natives from Tumbes. To everyone's surprise, these carried textiles, ceramic objects and some pieces of gold, silver and emeralds, making Ruiz's findings the central focus of this second expedition. Some natives were taken aboard Ruiz's ship to serve as interpreters.

When the new governor of Panama, Pedro de los Ríos, refused to allow for a third expedition to the south, the associates resolved for Pizarro to leave for Spain and appeal to the sovereign in person. Pizarro sailed from Panama for Spain in the spring of 1528, accompanied by Pedro de Candia, some natives and llamas, plus samples of fabric, gold and silver.

Pizzaro reached Seville in early summer. King Charles I, who was at Toledo, had an interview with Pizarro and heard of his expeditions in South America. The conquistador described the territory as rich in gold and silver that he and his followers had bravely explored "to extend the empire of Castile". The king, who was soon to leave for Italy, was impressed at his accounts and promised his support for the conquest of Peru. Queen Isabel, though, in the absence of the king, signed the Capitulación de Toledo on 6 July 1529, a license document that authorized Pizarro to proceed with the conquest of Peru. Pizarro was officially named the Governor, Captain general, Adelantado and Alguacil Mayor, of New Castile for the distance of 200 leagues along the newly discovered coast and invested with all authority and prerogatives, leaving his associates in secondary positions (a fact that later incensed Almagro and would lead to eventual discord). One of the grant conditions was that within six months, Pizarro should raise a sufficiently equipped force of 250 men, of whom 100 might be drawn from the colonies.

Pizarro could not raise the number of men the Capitulación required and sailed clandestinely from the port of Sanlúcar de Barrameda for the Canary Island of La Gomera in January 1530. He was there joined by his brother Hernando and the remaining men in two vessels that would sail back to Panama. Pizarro's third and final expedition left Panama for Peru on 27 December 1530.

In 1531, Pizarro once again landed in the coasts near Ecuador, the province of Coaque and the region of esmeraldas, where some gold, silver and emeralds were procured and then dispatched to Almagro. The latter had stayed in Panama to gather more recruits. Sebastián de Belalcázar soon arrived with 30 men. Though Pizarro's main objective was then to set sail and dock at Tumbes like his previous expedition, he was forced to confront the Punian natives in the Battle of Puná, leaving three or four Spaniards dead and many wounded. Soon after, Hernando de Soto, another conquistador who had joined the expedition, arrived with 100 volunteers and horses to aid Pizarro and with him sailed towards Tumbes, only to find the place deserted and destroyed. The two conquistadors expected that the settlers had disappeared or died under murky circumstances. The chiefs explained that the fierce tribes of Punians had attacked them and ransacked the place.

Atahualpa's refusal led Pizarro and his force to attack the Inca army in what became the Battle of Cajamarca on 16 November 1532. The Spanish were successful. Pizarro executed Atahualpa's 12-man honor guard and took the Inca captive at the so-called Ransom Room. By February 1533, Almagro had joined Pizarro in Cajamarca with an additional 150 men and 50 horses.

The Spanish sealed the conquest of Peru by entering Cuzco on 15 November 1533. Jauja, in the fertile Mantaro Valley, was established as Peru's provisional capital in April 1534, but it was high up in the mountains and too distant from the sea to serve as the capital. Pizarro founded the city of Lima on Peru's central coast on 6 January 1535, which he considered to be one of the most important things he had created in life.

By his marriage to N de Trujillo, Pizarro had a son also named Francisco, who married his relative Inés Pizarro, without issue. After Pizarro's death, Inés Yupanqui, whom he took as a mistress, favourite sister of Atahualpa, who had been given to Francisco in marriage by her brother, married a Spanish cavalier named Ampuero and left for Spain, taking her daughter who would later be legitimized by imperial decree. Francisca Pizarro Yupanqui eventually married her uncle Hernando Pizarro in Spain, on 10 October 1537; a third son of Pizarro who was never legitimized, Francisco, by Dona Angelina, a wife of Atahualpa that he had taken as a mistress, died shortly after reaching Spain.

Atahualpa's wife, 10-year-old Cuxirimay Ocllo Yupanqui, was with Atahualpa's army in Cajamarca and had stayed with him while he was imprisoned. Following his execution, she was taken to Cuzco and given the name Dona Angelina. By 1538, it was known she had borne Pizarro two sons, Juan and Francisco.

In Lima, on 26 June 1541 "a group of 20 heavily armed supporters of Diego de Almagro II "el mozo" stormed Pizarro's palace, assassinating him and then forced the terrified city council to appoint young Almagro as the new governor of Peru", according to Burkholder and Johnson. "Most of Pizarro's guests fled, but a few fought the intruders, numbered variously between seven and 25. While Pizarro struggled to buckle on his breastplate, his defenders, including his half-brother Martín de Alcántara, were killed". For his part, Pizarro killed two attackers and ran through a third. While trying to pull out his sword, he was stabbed in the throat, then fell to the floor where he was stabbed many times." Pizarro (who now was maybe as old as 70 years and at least 62), collapsed on the floor, alone, painted a cross in his own blood and cried for Jesus Christ. He died moments after. Diego de Almagro the younger was caught and executed the following year after losing the battle of Chupas.

Pizarro's remains were briefly interred in the cathedral courtyard; at some later time, his head and body were separated and buried in separate boxes underneath the floor of the cathedral. In 1892, in preparation for the anniversary of Columbus' discovery of the Americas, a body believed to be that of Pizarro was exhumed and put on display in a glass coffin. However, in 1977, men working on the cathedral's foundation discovered a lead box in a sealed niche, which bore the inscription "Here is the head of Don Francisco Pizarro Demarkes, Don Francisco Pizarro who discovered Peru and presented it to the crown of Castile." A team of forensic Scientists from the United States, led by Dr. william Maples, was invited to examine the two bodies and they soon determined that the body which had been honored in the glass case for nearly a century had been incorrectly identified. The skull within the lead box not only bore the marks of multiple sword blows, but the features bore a remarkable resemblance to portraits made of the man in life.

In the early 1930s, Sculptor Ramsey MacDonald created three copies of an anonymous European foot soldier resembling a conquistador with a helmet, wielding a sword and riding a horse. The first copy was offered to Mexico to represent Cortés, though it was rejected. The statue was taken to Lima in 1934 and re-purposed to represent Pizarro. One other copy of the statue resides in Wisconsin. (The mounted statue of Pizarro in the Plaza Mayor in Trujillo, Spain, was created by American Sculptor Charles Rumsey. It was presented to the city by his widow in 1926.)

The statue long stood an adjacent square to Peru's Government Palace. In 2003, after years of requests for the statue to be removed, the mayor of Lima, Luis Castañeda Lossio, approved the transfer of the statue to another location. Since 2004, however, Pizarro's statue has been in a park surrounded by the recently restored 17th-century walls in the Rímac District. The statue faces the Rímac River and the Government Palace.

After much wrangling between Pizarro and Almagro, it was decided that Pizarro would stay at a safer place, the Isla de Gallo, near the coast, while Almagro would return to Panama with Luque for more reinforcements – this time with proof of the gold they had found and the news of the discovery of the obviously wealthy land they had explored. The new governor of Panama, Pedro de los Ríos, had learned of the mishaps of Pizarro's expeditions and the deaths of various settlers who had gone with him. Fearing an unsuccessful outcome, he rejected Almagro's application for continued resources. In addition, he ordered two ships commanded by Juan Tafur to be sent immediately with the intention of bringing Pizarro and his crew back to Panama.

Pizarro is well known in Peru as the leader of the Spanish conquest. After his invasion, Pizarro destroyed the Inca state and while ruling the area for almost a decade, initiated the decline of local cultures. The Incas’ polytheistic religion was replaced by Christianity and much of the local population was reduced to serfdom under the Spanish elite. The cities of the Inca Empire were transformed into Spanish Catholic cities. Pizarro is also reviled for ordering Atahualpa's death despite the ransom payment (which Pizarro kept, after paying the Spanish king his due). Many Peruvians, including many of mainly indigenous descent, regard him negatively, although until relatively recently Pizarro had been portrayed positively, for instance in textbooks, for imposing Catholicism and creating a privileged class of mainly Spanish descent.