In 1960 the university was renamed Jan Evangelista Purkyně University after Jan Evangelista Purkyně, a Czech biologist. In 1990, following the Velvet Revolution it regained its original name. Since 1922, over 171,000 students have graduated from the university.

Masaryk University was founded on 28 January 1919 with four faculties: Law, Medicine, Science, and Arts. Tomáš Garrigue Masaryk, professor of Charles University and later the first president of Czechoslovakia, contributed greatly to the establishment of Masaryk University. (Masaryk in his scientific and political activities paid attention to the development of Czechoslovak universities and since the 1880s he emphasized the need for broad competition in scientific work. In this context, he pointed out that the only Czech university at that time needed a competitive institution for its development.) The founding of the second Czech university was possible only after the fall of the Austro-Hungarian monarchy because of the resistance of the German-controlled city council, which feared giving power to the Czech residents of Brno. Brno was at that time a bilingual city. A notable demonstration in favour of establishing a university in Brno happened in 1905.

From the beginning, the university suffered from a lack of money for development. The fragile state of public finances in 1923–1925 and 1933–1934 led to proposals to abolish both the Faculty of Arts and the Faculty of Science. Both faculties eventually survived until 17 November 1939 when the whole university was closed following the German occupation of Czechoslovakia. A number of professors of Masaryk University were executed or tortured; for example, the Faculty of Science lost one quarter of its teaching staff. Many of the executions took place in the Mauthausen concentration camp in 1942.