Hitzig, the son of a Berlin architect, was educated at the university there and obtained his MD in 1862. He was later appointed, in 1875, director of the Berghölzi asylum and professor of psychiatry at the University of Zurich. In 1885 Hitzig moved to similar posts at the University of Halle, posts he retained until his retirement in 1903.

In 1870, in collaboration with the German anatomist Gustav Fritsch (1838–1927), Hitzig published a fundamental paper, On the Excitability of the Cerebrum, which provided the first experimental evidence for cerebral localization. Following the important work of Pierre Flourens in 1824 it was widely accepted that, despite the discoveries of Paul Broca and John Neethlings Jackson, the cerebral hemispheres constituted a unity, the seat of intelligence, sensation, and volition and not the source of movement.

This was shown to be false when Hitzig and Fritsch electrically stimulated the cerebral cortex of a dog and elicited distinct muscular contractions. They identified five localized centers, which produced various movements on the side of the dog opposite to the side of the brain stimulated. Their work was soon confirmed by David Ferrier and opened up a vast research program, still, a century later, unfinished.

Hitzig himself continued with this work and in 1874 tried to define what soon became known as the motor area of the dog and the monkey. He also tried to identify, though less successfully, the site of intelligence, in the sense of abstract ideas, in the frontal lobes.