This study investigated the isomer-specific effects of cis-9,trans-11 (c9,t11) and trans-10,cis-12 (t10,c12) conjugated linoleic acid (CLA) on the metastasis of colon cancer cells in vitro and in vivo. Cell migration was examined by a Boyden chamber assay in SW480 cells. MMP-9 activity was monitored by gelatin zymography, and MMP-9 protein and mRNA levels were determined by Western blot and RT-PCR analysis, respectively, in SW480 cells. For the experimental metastasis, BALB/c mice were injected intravenously with CT-26 cells in the tail vein. Mice were fed a diet containing either no CLA or 0.1% c9,t11 or t10,c12 CLA for 4 weeks. In experimental metastasis, the numbers of pulmonary nodules were significantly lower in mice fed CLA isomers than in mice fed a control diet (P<.05). Results from the Boyden chamber assay revealed that c9,t11 CLA significantly inhibited cell migration (P<.05), whereas t10,c12 CLA had no effect on cell migration. The activity of MMP-9 was significantly inhibited by c9,t11 CLA (P<.05) but not by t10,c12 CLA. However, neither MMP-9 protein nor mRNA levels were altered by either of these CLA isomers. We have demonstrated that diets containing 0.1% c9,t11 and t10,c12 CLA were equally effective in inhibiting colon cancer cell metastasis in vivo. However, in vitro, only c9,t11 but not t10,c12 inhibited colon cancer cell migration and MMP-9 activity.