Fucosterol (2), oxygenated fucosterol, 24-ethylcholesta-4,24(28)-dien-3,6-dione (3), ${eta}$-sitosterol (1), and daucosterol (4) were isolated from ethyl acetate (EtOAc) fraction of Osmanthus fragrans var. aurantiacus flower by repeated silica gel, octadecyl silica gel, and Sephadex LH-20 column chromatography. The sterol structures were determined based on spectroscopic methods including nuclear magnetic resonance, infrared spectroscopy, fast atom bombardment mass spectrometry, and electron ionization mass spectrometry. Fucosterol (2), oxygenated fucosterol, and 24-ethylcholesta-4,24(28)-dien-3,6-dione (3) are representative sterols mainly isolated from marine algae, but rarely found in land plants. This is the first reported isolation of these sterols from O. fragrans var. aurantiacus. Interestingly, the oxygenated fucosterol (3) isolated inhibited the in vitro growth of the HCT-116 human colon cancer cell line with higher cytotoxic effects than doxorubicin, a well-known cytotoxic drug.