The founder of the modern-day Vatican Library was Pope Nicholas V (Tommaso Parentucelli). When he was elected in March 1447, the Vatican collection included about 350 Latin manuscripts, as well as a few Greek and Hebrew ones, which are described in an inventory produced under his predecessor Eugene IV. This patrimony, together with Nicolas V’s own books, constituted the beginnings of the collection, which Nicolas V generously expanded by prescribing that manuscripts be purchased in all the markets of Europe and of the East and that new ones be produced by a team of scribes. From the inventory made shortly after his death on 24th March 1455, and from other sources, it appears that he left more than 1,500 manuscripts, making the Pontifical collection the largest one in Europe at the time.

Some 20 years later, Sixtus IV (1471-1484) issued the papal bull Ad decorem militantis ecclesiae (15th June 1475), providing the Library with an endowment and with its first Librarian, Bartolomeo Platina. The number of manuscripts had increased to 2,527 by 1475 and to about 3,500 by 1481, when a new site was prepared for the library on the ground floor of the palazzo built by Nicholas III, with an entrance on the Cortile del Pappagallo, overlooking the cortile del Belvedere. It consisted of four rooms of unequal size which were called, respectively, Bibliotheca Latina, Bibliotheca Graeca (for works in these two languages); Bibliotheca Secreta (for manuscripts which were not directly accessible to readers, including some particularly precious codices); and Bibliotheca Pontificia (for Papal archives and registries). The Librarian was aided by three assistants and by a bookbinder. Reading was done on-site, in accordance with very strict regulations; but books were also loaned at that time, and the relative registers for the years 1475-1547 are still in existence (Vat. lat. 3964 and 3966).

In 1587, Sixtus V commissioned his architect Domenico Fontana to lay the foundations for a new, larger site for the Library. This building, which still houses the Library today, was constructed over the stairways which divided the Cortile del Belvedere and the courtyard which is now known as the Cortile della Biblioteca. On the top floor was a large room (70 m. by 15 m.) with two naves, which today is called the Salone Sistino and which was intended to contain the collections. Sixtus V issued precise rules for the use and the conservation of the collections.