King Philip IV of Spain authorized its founding. Cristóbal de Torres, the Archbishop of Santafé - colonial-era Bogotá - did the actual work, signing the founding documents and writing the university's original constitutions, which were then published by the chancellor Crístobal de Araque Ponce de León. Torres chose the Cross of Calatrava - a red Greek cross with ends shaped like fleurs-de-lis, associated with the medieval Spanish military order of the same name - as the university's symbol. He established its governing principle as Universitas Scholarium: a university from and for its students, not for its administrators. That principle still governs the institution in a recognizable form. The original constitutions, amended only modestly across four centuries, still set the terms of how students and faculty select leadership. In 1768 King Charles III formally recognized El Rosario as a Colegio Mayor, placing it alongside the six Spanish Colegios Mayores and tying its intellectual lineage explicitly to the University of Salamanca - the thousand-year-old mother of Spanish higher education.

Colombia's oldest university has not interrupted instruction once in its nearly four centuries of existence - except for a couple of years beginning in 1819. That was when General Pablo Morillo, the Spanish commander sent to reconquer Nueva Granada after the first wave of South American independence, used the Cloister as a prison. Many of the men who would go on to found the Colombian Republic - the alumni who had started the independence movement - were held there, in the same vaulted colonial rooms where they had taken their classes. Among those executed by the Spanish during this period were Camilo Torres Tenorio, Francisco José de Caldas y Tenorio, and Joaquín Camacho Lago, all El Rosario graduates, all later remembered as precursors and martyrs of independence. The university reopened after Colombian independence and has not closed since.

On 13 March 1762, José Celestino Mutis began teaching astronomy and mathematics at El Rosario. Mutis was a Spanish-born botanist, astronomer, and physician who had come to Nueva Granada as the personal doctor of a viceroy and stayed for the rest of his life. At El Rosario he taught for more than five years - until 1767 - lecturing on mathematics and on physical and natural sciences, both in Latin. He is buried in the university's chapel. Beyond the classroom, Mutis led the Royal Botanical Expedition of the New Kingdom of Granada from 1783 onward, producing the greatest single botanical survey of the Spanish Empire: more than 6,000 watercolor illustrations, many by Indigenous and mestizo artists Mutis trained, documenting the plants of what is now Colombia and northern Ecuador. His portrait on the old 200-peso banknote, paired with the image of El Rosario's cloister, connected the university to the intellectual project that gave Colombia its first scientific mapping of its own living country. In 1790 El Rosario established the first medical school in Colombia under Dr. Vincent Cancino Román.

The Cloister - El Claustro - is the main campus, on 14th Street in the historic center of Bogotá, separated from Avenida Jiménez de Quesada by the Plazoleta del Rosario. The building is seventeenth-century Spanish colonial architecture in stone and adobe, built by Fray Cristóbal de Torres himself during the university's founding. Experts consider it the oldest building of its type in the city. In 1909 the Catalan sculptor Renart Dionisio García cast a bronze statue of the founder, which stands inside the cloister today. The Aula Máxima - the Great Hall, where protocolary ceremonies are held - contains an art collection dating from around 1700, considered one of the best small galleries in Colombia. The Cloister carries the designation National Architectural and Cultural Heritage. Its Historic Archive preserves documents of the university and, by extension, of Colombia itself: the oldest is from 1646, a letter from King Philip IV of Spain to Archbishop Torres responding to the archbishop's request to establish the institution.

The list of graduates who have shaped Colombian public life is remarkable for both its length and its continuity. Presidents: Carlos Holguín Mallarino, Ramón González Valencia, Miguel Abadía Méndez, Darío Echandía, Eduardo Santos, Alberto Lleras Camargo, Alfonso López Michelsen, Andrés Pastrana - to name only some who served in the twentieth century. Vice President Germán Vargas Lleras. Former Bogotá mayor Samuel Moreno Rojas. The Liberal Party leader Rafael Uribe Uribe. Constitutional Court justices and presidents. Attorneys general. Ambassadors. Ministers of defense, environment, and government. The university's influence extended into the 1886 Constitution's Supreme Court - the so-called Golden Court of 1936 - which was composed in its majority of graduates of El Rosario's Faculty of Jurisprudence. The associated all-male secondary school, which operated until 2008 and shared the university's name, added its own list of distinguished alumni, including the poet and novelist Álvaro Mutis and the journalist Daniel Coronell. El Rosario drafted the 1991 Constitution's reform; it drafted the nineteenth-century independence; it will almost certainly have a hand in whatever Colombia becomes next.