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A New Yorker by birth, Hofstadter graduated from the College of the City of New York in 1935 and gained his MA and PhD at Princeton University in 1938. From 1939 he held a fellowship at the University of Pennsylvania, and in 1941 returned to the College of the City of New York as an instructor in physics. From 1943 to 1946 Hofstadter worked at the Norden Laboratory Corporation, and from there took on an assistant professorship in physics at Princeton University. In 1950 he moved to Standford University as an associate professor and was made full professor in 1954.

His early research was in the fields of infrared spectroscopy, the hydrogen bond, and photoconductivity. One of his first notable achievements, in 1948, was the invention of a scintillation counter using sodium iodide activated with thallium. He is noted for his studies of the atomic nucleus, for which he received the 1961 Nobel Prize for physics (shared with Rudolph Mössbauer).

At Stanford, Hofstadter used the linear accelerator to study the scattering effects of high electrons fired at atomic nuclei. In many ways these experiments were similar in concept to Rutherford's original scattering experiments. He found that the distribution of charge density in the nucleus was constant in the core, and then decreased sharply at a peripheral region. The radial distribution of charge was found to vary in a mathematical relationship that depended upon the nuclear mass. Further, Hofstadter was able to show that nucleons (protons and neutrons) were not simply point particles, but had definite size and form. Both appeared to be composed of charged mesonic clouds (or shells) with the charges adding together in the proton, but canceling each other out in the neutral neutron. This led him to predict the existence of the rho-meson and omega-meson, which were later detected.

Hofstadter served as director of the high-energy physics laboratory at Stanford from 1967 to 1974.