Objective: To investigate the effect of ω-3 fatty acids on the proliferation and gene expressions of vascular endothelial growth factor (VEGF), syndecan-1 and Ki-67 of SW480 colon cancer stem cells. Methods: The CD133+/CD44+/ESA+ cell subgroups (SW480 colon cancer stem cells) in the SW480 colon cancer cells were sorted by flow cytometry. Then, the colon cancer cells were treated with serial concentrations (15, 30, 60 μg/μL) of EPA (eicosapentaenoic acid) or serial concentrations (15, 30, 60 μg/μL) of DHA (docose hexaenoie acid), or vehicle (control), and the cell proliferations of each cell group were determined by MTT assay. Furthermore, the gene expressions of VEGF, syndecan-1 and Ki-67 in colon cancer cells after treatment of EPA (60 μg/μL) or DHA (60 μg/μL) were analyzed by RT-PCR method. Results: Both low concentrations of EPA and DHA had no noticeable effect on the growth of colon cancer cells (both P<0.05), while either medium or high concentration (30 and 60 μg/μL) of EPA and DHA significantly inhibited the growth of colon cancer cells, which had statistical differences compared with control group (all P<0.05). Both EPA and DHA at a concentration of 60 μg/μL could significantly suppress the gene expression of syndecan-1 and Ki-67 (both P<0.05), but showed no evident effect on VEGF gene expression (both P>0.05). Conclusion: ω-3 fatty acids can inhibit the proliferation of SW480 colon cancer stem cells, and the mechanism is probably associated with its suppression of syndecan-1and Ki-67 expression.