Born at Corteno near Brescia (now in Italy), Golgi studied medicine at Pavia University and thereafter mainly concerned himself with research on cells and tissues. In 1873, while serving as physician at the home for incurables, Abbiategrasso, he devised a method of staining cells by means of silver salts. This allowed the fine processes of nerve cells to be distinguished in greater detail than before and enabled Golgi to confirm Wilhelm von Waldeyer's view that nerve cells do not touch but are separated by gaps called synapses. Golgi also found a specialized type of nerve cell, later called the Golgi cell, which, by means of fingerlike projections (dendrites), serves to connect many other nerve cells. This discovery led to the formulation (by Waldeyer) and establishment (by Santiago Ramón y Cajal) of the neuron theory – a theory that Golgi was nevertheless strongly opposed to.

Golgi was also the first to draw attention to the Golgi bodies: flattened cavities parallel to the cell's nuclear membrane whose function appears to be packaging and exporting various materials from the cell. Apart from work on the sense organs, muscles, and glands, Golgi studied varying forms of malaria. He found that different species of the protozoan parasite Plasmodium are responsible for the two types of intermittent fever – the tertian and quartan. He also established that the onset of fever coincides with the release into the blood of the parasitic spores from the red blood cells.

Golgi served as professor of histology (1876) and then of general pathology (1881) at Pavia University. In 1906 he shared with Ramón y Cajal the Nobel Prize for physiology or medicine for his work on the structure of the human nervous system.