Pickering, the younger brother of Edward Pickering, was also an astronomer. Born in Boston, Massachusetts, he studied at the Massachusetts Institute of Technology where he worked after graduating in 1879. In 1887 he moved to the Harvard College Observatory where his brother was director. He set up a number of observing stations for Harvard including that at Arequipa, Peru, in 1891 and Mandeville, Jamaica, in 1900. He took charge of the latter in 1911, converting it into his own private observatory following his retirement in 1924.

He also helped Percival Lowell set up his private observatory in Flagstaff, Arizona, and, also like Lowell, concerned himself with the trans-Neptunian planet. In 1919, on the basis of past records, he predicted that a new planet would be found near the constellation of Gemini but photographic surveys failed to confirm his prediction. When the planet was finally detected in 1930 by Clyde Tombaugh, Pickering made a somewhat exaggerated claim to be its discoverer.

He made extensive observations of Mars and claimed, like Lowell, that he saw signs of life on the planet by observing what he took to be oases in 1892. He went further than Lowell however when in 1903 he claimed to observe signs of life on the Moon. By comparing descriptions of the Moon from Giovanni Riccioli's 1651 chart onward, he thought he had detected changes that could have been due to the growth and decay of vegetation.

He was more successful in 1899 when he discovered Phoebe, the ninth satellite of Saturn. This was the first planetary satellite with retrograde motion to be detected, i.e., with orbital motion directed in an opposite sense to that of the planets. His 1905 report of a tenth satellite, which he confidently named Themis, was not substantiated.