Polyunsaturated fatty acids (PUFA) regulate various biological functions and are involved in a variety of diseases. Eicosapentaenoic acid (20:5, EPA), one of the omega-3 PUFA, has been reported a number of actions including suppression of platelet aggregability and decrease in serum lipids and is well known to be useful in preventing the atherosclerotic diseases. In this paper, we demonstrated that highly purified ethyl eicosapentaenoate (EPA-E) prevents cholesterol (CH) gallstone formation in hamster model. Repeated administration of EPA-E to animals fed a lithogenic diet for 6 weeks decreased the incidences of both CH crystal and CH gallstone formations in gallbladder bile. Contrary, bezafibrate, one of fibric acid derivative which were reported to increase CH saturation index (CSI) in bile, significantly increased the incidences of CH crystal and gallstone formations. EPA-E did not affect the CSI, but markedly increased biliary phospholipid concentration. In the same model, ethyl palmitate (16:0), ethyl oleate (18:1), ethyl linolate (18:2, omega-6) and ethyl arachidonate (20:4, omega-6) had no effects both on biliary lipids composition and CH gallstone formation. These result suggest the benefit of EPA-E in the prevention of CH gallstone.