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N-3 polyunsaturated fatty acids (N-3 PUFAs) have been shown to inhibit the induction and progression of many kinds of tumors, and increase the therapeutic effects of numerous chemotherapeutics,but the anticancer effect on cancer stem cells (CSCs) of Colorectal cancer (CRC) is not described before.In the present study, we cultivated stem like spheres from SW620 cell line in the specific serum-free medium and evaluated the spheres features by immunofluorescence, soft afar clone formation, Cell cycle distribution, and then discovered the spheres generated from SW620 cells dedifferentiation by RT-PCR analysis of pluripoteney genes, such as Sox-2, Oct-4, Bmi-1, finally proved the spheres were CSCs through differentiation assay.To explore the use of N-3 PUFAs on CSCs, the spheres were treated with N-3 PUFA (DHA/EPA), and we found treatment of the spheres with DHA or EPA or together for 72 hours led to apoptosis and resulted in a progressive loss of viability, DNA fragmentation, and an increase in Annexin V expression.Both DHA and EPA can enchancing chemotherapy sensitivity effect of Mitomycin C, especially DHA.Taken together, these results provided evidences that N-3 PUFAs exerted a direct anticancer action,which might attribute to its antiproliferative and pro-apoptotic effect on CSCs.