Both conjugated linoleic acid (CLA) and eicosapentaenoic acid (EPA) have an antitumor effect. Hence, we hypothesized that a combination of conjugated double bonds and an (n-3) highly unsaturated fatty acid would produce stronger bioactivity. To verify the antitumor effect of conjugated EPA (CEPA), we transplanted DLD-1 human colon tumor cells into nude mice, and compared the tumor growth between CEPA-fed mice and CLA- and EPA-fed mice. After tumor cell inoculation, mice were assigned to 1 of 4 groups (control, CLA, EPA, and CEPA) consisting of 10 mice each. The control group received only safflower oil fatty acids, whereas the remaining groups received a mixture of safflower oil fatty acids and 20 g/100 g of total fatty acids as CLA, EPA, or CEPA. Mice were fed once every 2 d for 4 wk at a dose of 50 mg/mouse at each feeding. After 4 wk, tumor growth in CEPA-fed mice was significantly suppressed, compared with that in CLA- (P < 0.005) and EPA-fed mice (P < 0.001). DNA fragmentation in the tumor tissues of the CEPA-fed mice occurred more frequently than in the CLA- (P < 0.001) and EPA-fed mice (P < 0.001), suggesting that CEPA induced apoptosis in the tumor tissues. To further investigate the mechanism, the level of oxidative stress in the tumor tissues was determined. The CEPA-fed mice showed significant lipid peroxidation, compared with the CLA- (P < 0.001) and EPA-fed mice (P < 0.001). Therefore, we verified that CEPA has a stronger in vivo antitumor effect than EPA and CLA, and that CEPA acts through induction of apoptosis via lipid peroxidation.