This study is to evaluate the effect of dietary omega-3 polyunsaturated fatty acid (omega-3 PUFA) and/or calcium supplementation on colon tissue of the carcinogenic N-methyl-N-nitrosurea (NMU)-injected rats and to investigate this effect by the assessment of the oxidative stress. The rats were divided into four groups: those fed with a standard diet, with a diet supplemented by omega-3 PUFA, those fed with a diet with calcium, and those fed with a standard diet with the combination of omega-3 PUFA and calcium. Rats were injected with an intrarectal NMU. After 32 weeks, colon tissue specimens and plasma were taken to histopathologically investigate and analyze tissue superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) activities and erythrocyte MDA levels. The tumor incidences in supplemented-diet groups II and IV were found to be significantly lower when compared with those of the controls (P < 0.05). Superoxide dismutase and glutathione peroxidase antioxidative enzyme activities in colorectal tissue were increased in the study groups when compared with control rats (P < 0.001) and MDA levels were significantly lower than in the controls (P < 0.001) while the levels in group IV were rather decreased than those in group III (P = 0.011). These results suggest that the dietary supplementation of PUFA and/or calcium may be useful in the prevention of colorectal tumor formation.