Unraveling the role of microRNAs in cancer has gained growing interest in the last few years. In this study, we identified miR-215 as a potential prognostic biomarker and miR-129 as a potential tumor suppressor and a novel therapeutic target in colorectal cancer. We showed that miR-215 levels were significantly decreased in tumors compared with corresponding normal tissues in patients diagnosed with stage II or stage III colorectal cancer. Furthermore, we described a novel mechanism mediated by miR-129 to trigger apoptosis by suppressing a key anti-apoptotic protein, BCL2. The expression of miR-129 was significantly and progressively down regulated in colorectal cancer tissue specimens compared with the adenomas and normal control samples. More importantly, we demonstrated that miR-129 increased the cytotoxic effect of 5- fluorouracil, in vitro and in vivo. We expect that our findings will provide a better insight into the role of microRNAs in cancer and will allow us to carry these molecules into clinical application.