Born into a poor family, Huntington worked as an itinerant peddler and became a prosperous merchant in Oneonta, N.Y., before moving to Sacramento, Calif., in the gold rush year of 1849. There he became a partner with Mark Hopkins in a successful wholesale-retail firm that specialized in miners' supplies. In the late 1850s he became interested in a plan to link California with the eastern United States by rail, and he joined Hopkins, Leland Stanford, and Charles Crocker (a group later known as the Big Four) in their joint incorporation of the Central Pacific Railroad in 1861, which was soon designated as the western part of the projected transcontinental railroad.

During the actual construction (1863–69) of the Central Pacific Railroad, Huntington lobbied for the company in the east, working to secure financing and favourable legislation from Congress and the federal government. In 1865 the Big Four formed the Southern Pacific Railroad, which constructed rail lines down southern California and across the Southwest to New Orleans, again with Huntington serving as chief political and financial lobbyist. Huntington helped expand the Southern Pacific Railroad into a 9,600-mile (15,450-kilometre) line that was the foundation of rail service in California and the western link for a southern transcontinental rail route. He also extended the lines of the Chesapeake and Ohio Railway, which he had bought in 1869, to link with the Southern Pacific, forming a 4,000-mile (6,440-kilometre) continuous track from San Francisco to Newport News, Va.

Huntington became president of the Southern Pacific–Central Pacific rail system in 1890 and thus controlled a vast empire of rail lines and agricultural landholdings in California until his death.