The son of a prominent radiologist, Edward Chamberlain, Owen Chamberlain followed his father's interest in physics. Born in San Francisco, he graduated from Dartmouth College in 1941 and gained his doctorate in physics from the University of Chicago in 1949. From 1948 until 1950 he was an instructor in physics at the University of California at Berkeley, becoming associate professor in 1954, professor in 1958, and emeritus professor from 1989.

The onset of America's involvement in World War II interrupted his university studies, and he spent the years 1942–1946 under the leadership of Emilio Segrè; working on the Manhattan atom-bomb project at Los Alamos. There he investigated spontaneous fission of the heavy elements and nuclear cross sections. Later he worked with Enrico Fermi on neutron diffraction by liquids.

At Berkeley, Chamberlain experimented with the bevatron particle accelerator of the Lawrence Radiation Laboratory, and in 1955 (together with Segrè, C. Weigand, and T. Ypsilantis) discovered the antiproton – a particle with the same mass as the proton, but of opposite (negative) charge. For their discovery, Chamberlain and Segrè received the 1959 Nobel Prize for physics. The existence of antiparticles had been predicted by Paul Dirac's theory of 1926, and the first of these, the positive electron (or positron) had been found by Carl David Anderson in cosmic radiation in 1931.

Chamberlain's later work was on the interaction of antiprotons with hydrogen and deuterium, the production of antineutrons from antiprotons, and the scattering of pions.