After graduation from Beloit College in 1866, Chamberlin worked for two years as a teacher and later principal in a high school near Beloit. He was married to Alma Wilson in 1867.
In 1868–1869, Chamberlin spent a year taking graduate courses, including geology, at the University of Michigan to strengthen his scientific background. Subsequently (1869-1873) he became professor of natural science at the State Normal School in Whitewater, Wisconsin. He joined the Beloit faculty in 1873, where he was professor of geology, zoology, and botany. In 1873 he also became one of several part-time participants in conducting a comprehensive geological survey of Wisconsin. His geologic mapping work in southeastern Wisconsin, a region mantled with thick glacial deposits, led him to recognize multiple episodes of glaciation during the Pleistocene. His terminology for glacial stages in North America is still in use, with minor modifications.
In 1876 Chamberlin became chief geologist for the Wisconsin geological survey, supervising the completion of the survey and the publication of the four-volume report, for which he authored sections on glacial deposits, Paleozoic and Precambrian bedrock geology, lead-zinc ore deposits, artesian wells, and soils. The project brought him national attention and led to his appointment as head of the glacial division of the US Geological Survey in 1881. He later was president of the University of Wisconsin (1887 - 1892).
In 1892 he accepted the offer to organize a department of geology at the new University of Chicago, where he remained as a professor until 1918). From 1898 to 1914 he was president of the Chicago Academy of Sciences.
He developed the planetesimal theory, which states that Earth was made from smaller objects that gradually built the planets by accretion. From this theory and other geological evidence he concluded that Earth was much older than assumed by Lord Kelvin (ca 100 million years) at the time.
His papers are housed in the Beloit College archives, along with the papers of his son, Rollin T. Chamberlin, who was also a geologist.. There are buildings named for him on the Beloit College and University of Wisconsin-Madison campuses. The lunar crater Chamberlin and a crater on Mars are named in his honor.