His first documented appearance is in 1560 as a tenor at the Hofkapelle in Prague under Habsburg ruler Archduke Maximilian; Regnart claimed to have worked there since 1557.
The first fruits of these studies, Il primo libro delle canzone italiane, would be published in 1574, with many subsequent volumes to follow.
It was in the 1570s that his volumes of three-voice Teutsche Lieder (German songs) appeared, printed by the Gerlachs of Nuremberg; they sold very well, were reprinted several times, and were arranged in tablature by several composers.
On New Year's Day 1585, Regnart became Kapellmeister, and revamped the court's musical activities to great success, hiring many new Dutch and Italian singers and becoming quite wealthy himself.
Regnart's works were regularly anthologized well into the 17th century, and his music was held in high regard by such theorists as Michael Praetorius and Jacob Burmeister.