At the University of Padua (1538), Colombo studied medicine and surgery and succeeded Andreas Vesalius as professor of surgery in 1543. In 1546 Colombo became the first professor of anatomy at the University of Pisa and, in a similar position at the Sapienza, or Papal University, in Rome (1548–59), was surgeon to Pope Julius III.

De re anatomica (1559; “On Things Anatomical”), his only formal written work, includes several important original observations derived from his dissections on both living animals and human cadavers. His descriptions of the mediastinum (organs and tissues within the thoracic cavity, excluding the lungs), pleura (membrane surrounding the lungs), and peritoneum (membrane surrounding the abdominal organs) are the best made to that time. Most important, however, was his description of general heart action, which correctly stated that blood is received into the ventricles during diastole, or relaxation of the heart muscle, and expelled from them during systole, or contraction. He clearly outlined circulation of venous blood from the right ventricle, through the pulmonary artery to the lungs, whence it emerges bright red after mixture with a “spirit” in the air, and returns to the left ventricle through the pulmonary vein.

Although pulmonary circulation had been espoused clearly as early as the 13th century, accounts of it were unknown to contemporary science, and Colombo was recognized by his colleagues and by William Harvey as the discoverer of the phenomenon.