Objective: To investigate the influence of n-3 polyunsaturated fatty acids (n-3 PUFAs) on colorectal tumor formation induced by N-methyl-N-nitrosourea (MNU) in rats and the mechanism. Methods: Sixty SD rats were equally randomized into experimental group and control group. Rats in both groups underwent administration of MNU by enema to induce colorectal cancer, and simultaneously, those in experimental group were gavaged with n-3 PUFAs, while those in control group were administered the same volume of saline in the same fashion. Sixteen weeks later, the general conditions between the two groups of rats were compared, after that, rats were sacrificed, the tumor occurrence and pathological features of the tumors were observed, the concentration of n-3 PUFAs in erythrocyte membrane was measured by gas chromatography, and global methylation level in peripheral blood cells was determined by liquid chromatography-mass spectrometry. Results: The incidence of hematochezia was lower, and food-intake as well as body weight gain were greater in experimental group than those in control group (all P<0.05). Colorectal tumor formation was found in both group of rats and all tumors were identified as adenocarcinoma, but the tumor formation rate in experimental group was significantly lower than that in control group (63.33% vs. 86.67%, P<0.05), with reduced tumor size and multiple lesions. The concentration of n-3 PUFAs in erythrocyte membrane and global methylation level in peripheral blood cells in experimental group were significantly higher than those in control group (both P<0.05). Conclusion: n-3 PUFAs can effectively inhibit the occurrence of colorectal cancer induced by MNU in rats, which may probably be associated with their promotion of DNA methylation.