Juan de Céspedes was born in 1501 or 1505 in Argamasilla de Calatrava, Castile-La Mancha in a family of hidalgos from Toledo. His parents were Lope de Céspedes and María Ruiz and he had one brother; Diego. In 1521, he left Spain for Santo Domingo, Hispaniola, the first stronghold of the Spanish in the Americas. De Céspedes accompanied Rodrigo de Bastidas in the conquest of the Tairona and foundation of Santa Marta in 1525. After the Spanish kingCarlos V installed García de Lerma as governor of Santa Marta on December 20, 1527, the latter named Juan de Céspedes Captain of the Infantry and sent him on an expedition into the Valle-Dupar, Pocigüeyca, a village south of Santa Marta towards Ciénaga, and the Magdalena River. After this expedition that took two years, De Céspedes was sent by Pedro Fernández de Lugo into the interior of the province of Santa Marta. When in 1536 Gonzalo Jiménez de Quesada organised the main expedition into the Colombian Andes, he named De Céspedes as one of eight captains; of cavalry. At this expedition also his brother Hernando de Prado joined. In the lower parts of the Magdalena River, at the confluence with the San Jorge and Cauca Rivers, Juan de Céspedes and Juan de Sanct Martín reached first and were joined by the other troops of De Quesada. After the journey of a year into the Muisca Confederation, the troops settled on the Bogotá savanna. De Céspedes was sent south, while the other troops went north, into the territories of the zaque of Hunza. De Céspedes reached Fusagasugá in May 1537 with forty infantry and 15 horses. On July 15, 1537, Juan de Céspedes conquered and founded Pasca, Cundinamarca. De Céspedes continued south and west from there through the Sumapaz Páramo into the territories of the Sutagao. He was accompanied by Juan López de Herrera and suffered from the cold climate. As a result of his bravery during this harsh conquest, De Céspedes was awarded the encomiendas of Ubaque, Chipaque, Quetame and Subachoque, to the east of and on the Altiplano Cundiboyacense. The encomiendas of Ubaque and Quetame were passed on to his son Lope de Céspedes.
In 1539, the brother of the founder of Bogotá, Gonzalo Jiménez de Quesada, Hernán Pérez de Quesada had taken the governance of the New Kingdom of Granada. In this year, the conquistadors, among which De Céspedes, asked the King of Spain to construct a hospital in Bogotá. Under the command of Pérez de Quesada, De Céspedes and Juan de Sanct Martín tortured the last zipa of the Muisca, Sagipa, by cutting and burning the soles of the feet of the Muisca ruler. De Céspedes was sent westward into the terrain of the Panche, who were beaten in the Battle of Tocarema in August 1538. De Céspedes passed through San Antonio del Tequendama. From 1542 to 1543, De Céspedes was mayor of Bogotá, the capital of New Kingdom of Granada. He succeeded Hernán Venegas Carrillo, who occupied this position after De Céspedes again. In 1543, Alonso Luis de Lugo sent De Céspedes back to Santa Marta to help rebuild the city that was destroyed and burned by the French pirateRoberto Baal. From Santa Marta, De Céspedes participated in conquests in the interior. In 1546, De Céspedes returned to the capital in the Andes and served a second term as mayor of the city. The first president of the New Kingdom of Granada, Andrés Díaz Venero de Leyva, named Juan de Céspedes his lieutenant general, a position he held until it was abolished in 1570. In 1565, De Céspedes constructed the Santa Bárbara church in Bogotá, that still exists today. He constructed the chapel thanking the Saint of Thunder, as he escaped death of a lightning strike at that place. The lightning strike did kill one of his slaves. Juan de Céspedes had one son with an indigenous woman named Isabel. De Céspedes married Isabel Romero, one of the first Spanish women who arrived at the Spanish colony in northern South America, the widow of soldier Juan Francisco Lorenzo, who drowned in the Opón River. The marriage of De Céspedes and Romero was the first wedding in the New Kingdom of Granada, together with Lope Rioja and Elvira Gutiérrez. The couple had two sons, Antonio and Lope, and De Céspedes one stepdaughter; María, daughter of Isabel Romero and her first husband who was given the last name of Juan. Lope de Céspedes served as mayor of Bogotá from 1577 to 1578 and in 1605 and Antonio held the same position from 1591 to 1592 and 1596 to 1597. Juan de Céspedes died in late 1573 or 1576 in the New Kingdom of Granada. The house of De Céspedes was discovered on the site of the first convent of San Águstin, when it was moved to the San Francisco church, the oldest remaining church in Bogotá.