We have previously shown that conjugated eicosapentaenoic acid (CEPA), which is prepared by alkaline treatment of eicosapentaenoic acid and contains conjugated double bonds, suppresses tumor growth in vivo. In this earlier study, blood vessels were observed on the tumor surface in control mice, whereas in CEPA-treated mice, no such vessels were observed and the inner part of the tumor was discolored. These observations suggest that CEPA might suppress cancer cell growth through malnutrition due to a suppressive effect on tumor angiogenesis. In this study, the antiangiogenic effects of CEPA were investigated in vitro. CEPA at 5 μmol/L inhibited vascular endothelial growth factor (VEGF)-stimulated tube formation by human umbilical vein endothelial cells (HUVEC) (P < 0.05) and also inhibited VEGF-stimulated migration of HUVEC at a concentration of CEPA that suppressed tube formation (P < 0.05) but did not influence cell proliferation. The antiangiogenic mechanism of CEPA was investigated in vitro by measuring the secretion and expression of well-characterized angiogenic factors associated with cell migration, such as matrix metalloproteinases (MMP). CEPA at a concentration that suppressed tube formation inhibited secretion and mRNA expression of MMP2 and MMP9 in VEGF-stimulated HUVEC (P < 0.05). Our findings suggest that CEPA has potential use as a therapeutic dietary supplement for minimizing tumor angiogenesis.