It was founded in 1544 as the world's second Protestant academy (after the University of Marburg) by Duke Albert of Prussia and charted by the King Sigismund II Augustus.
Albert, former Grand Master of the Teutonic Order and first Duke of Prussia since 1525, had purchased a piece of land behind Königsberg Cathedral on the Kneiphof island of the Pregel River from the Samland chapter, where he had an academic gymnasium (school) erected in 1542.
The newly established Protestant duchy was a fiefdom of the Crown of the Kingdom of Poland and the university served as a Lutheran counterpart to the Catholic Kraków Academy.
Since the Prussian lands lay beyond the borders of the Holy Roman Empire, both Emperor Charles V and Pope Paul III withheld their approval, nevertheless the Königsberg academy received the royal privilege by King Sigismund II Augustus of Poland on 28 March 1560.
Subsequent rectors included numerous Hohenzollern Prussian royals (at last Crown Prince William 1908–1918), who had never been to the university, usually represented by a prorector in charge of academic affairs.
The Prussian lands remained unharmed by the disastrous Thirty Years' War, which gained the Königsberg university an increasing popularity among students.
Large numbers of Petrine officials trooped the university to cameralist theory and administrative practices which thus shaped Russia's government.
It was later associated with the names of Hermann Minkowski (Albert Einstein's teacher), Adolf Hurwitz, Ferdinand von Lindemann and David Hilbert, who was one of the greatest modern mathematicians.
The mathematicians Alfred Clebsch and Carl Gottfried Neumann (both born in Königsberg and educated under Ludwig Otto Hesse) founded the Mathematische Annalen in 1868, which soon became the most influential mathematical journal of the time.
The Senate Hall contained a portrait of Emperor Frederick III by Lauchert and a bust of Immanuel Kant by Hagemann, a student of Schadow.
When General Otto Lasch signed the capitulation on 9 April, the historic inner city was destroyed by the attacks, and 80% of the university campus lay in ruins.