Around 1850 Flagler became a grain merchant in Bellevue, Ohio, where he met Rockefeller and sold grain through him. With $50,000 capital, Flagler made an unsuccessful attempt to manufacture salt in Michigan and returned to Cleveland, where in 1867 he joined Rockefeller in an oil company that became Standard Oil in 1870. Active in the development of that corporation, he served as director of Standard Oil of New Jersey until 1911.

In 1883 Flagler visited Florida and three years later purchased several railway lines that he combined as the Florida East Coast Railway. During the 1890s he built a chain of luxury hotels along the rail line as well as in Nassau, in the Bahama Islands. He also dredged Miami harbour and established steamship lines to Key West and Nassau.