Interested in astronomy from an early age, Keeler became assistant to the noted astronomer Samuel P. Langley at the Allegheny Observatory, Pittsburgh, Pa., in 1881. From 1886 to 1891 he was on the staff of the Lick Observatory, Mount Hamilton, Calif., where he headed the spectroscopic program. Among the outstanding results that he achieved was the measurement of the rate of expansion of the Orion Nebula together with proof that it is located within the Milky Way Galaxy.

In 1891 Keeler became director of the Allegheny Observatory, where he performed his studies of Saturn's (Saturn) rings. Returning to the Lick Observatory as director in 1898, he undertook photographic observations of 120,000 galaxies with the recently acquired Crossley 36-inch (91-centimetre) reflecting telescope. This work established the reflecting telescope as the supreme instrument for photographing faint celestial objects and demonstrated that a spiral galaxy is the most common type of galaxy in the observable universe.