Maestlin studied theology, mathematics, and astronomy / astrology at the Tübinger Stift in Tübingen, a town in Württemberg. He graduated as Magister in 1571 and became in 1576 a Lutheran deacon in Backnang, continuing his studies there.

In 1580 he became a Professor of mathematics, first at the University of Heidelberg, then at the University of Tübingen were he taught for 47 years from 1583. In 1582 Maestlin wrote a popular introduction to astronomy.

Among his students was Johannes Kepler (1571 - 1630). Although he primarily taught the traditional geocentric Ptolemaic view of the solar system, Maestlin was also one of the first to accept and teach the heliocentric Copernican view. Maestlin corresponded with Kepler frequently and played a sizable part in his adoption of the Copernican system. Galileo Galilei's adoption of heliocentrism was also attributed to Maestlin.

The first known calculation of the (inverse) golden ratio as a decimal of "about 0.6180340" was written in 1597 by Maestlin in a letter to Kepler.

• Catalogued the Pleiades cluster on 24 December 1579. Eleven stars in the cluster were recorded by Maestlin, and possibly as many as fourteen were observed.
• Occultation of Mars by Venus on 3 October 1590, seen by Maestlin at Heidelberg.

• Asteroid 11771 Maestlin, discovered in 1973
• Lunar Crater Maestlin
• Lunar Rille Rimae Maestlin