[Background] The antitumor mechanism of ω-3 polyunsaturated fatty acids represented by DHA and EPA has not been fully investigated. [Objective] To explore the relationship among ω-3 polyunsaturated fatty acids, Fusobacterium nucleatum and colorectal cancer. [Methods] After the suppressive effect of ω-3 polyunsaturated fatty acids (DHA, EPA, ALA) on human colon cancer cell line, Caco-2, and normal colon epithelium cell line, NCM460, was assayed, we investigated the impact of these ω-3 polyunsaturated fatty acids on F. nucleatum, including growth, adhesive ability to Caco-2 cells, and the expression of virulence genes such as Fap2, FadA and RadD. [Results] After treated with 30 μg/ml DHA, EPA or ALA respectively, the growth of Caco-2 cells were suppressed 9.09%, 4.95% and 7.52% correspondingly, meanwhile the growth of NCM460 cells were suppressed 31.15%, 25.48%, 29.11%, and only dose-dependent effect was identified. After treated with 30 μg/ml DHA, EPA and ALA for 12 hours, the adhesive ability of F. nucleatum to Caco-2 cells was inhibited by 81.04% (P=0), 93.63% (P=0) and 68.63% (P=0) respectively, which was consistent with the transcriptive level assay results of FadA, Fap2 in F. nucleatum. The expression of Fap2 was considerably suppressed by 10.22% (P=0.027) after 30 μg/mL DHA treatment; FadA, Fap2 were markedly suppressed by 23.49% (P=0) and 15.09% (P=0.003) by 30 μg/mL EPA; and for 30 μg/mL ALA treatment, FadA was significantly suppressed by 26.75% (P=0.012). [Conclusion] Based on our above results and previous reports that DHA, EPA and ALA only inhibited the growth of F. nucleatum temporally, we proposed that ω-3 polyunsaturated fatty acids i-e EPA and DHA significantly attenuated the adhesive ability of F. nucleatum to host cells via suppressing the expression of adhesion-related genes such as FadA, Fap2, which made major contribution to their antitumor activity, rather than inhibiting the growth of tumor cells and F. nucleatum directly. The effects and mechanisms of ω-3 polyunsaturated fatty acids i-e DHA and EPA on gut bacterium i-e F. nucleatum that play important roles in the initiation and progression of colorectal tumors deserve further research.