Recent evidence shows that altered microRNA-126 (miR-126) expression is implicated in the progression of colorectal cancer (CRC). However, the precise roles and mechanisms of miR-126 in CRC remain unclear. The aim of this study was to investigate the roles of miR-126 in CRC cells and to elucidate miR-126-mediated mechanisms in CRC cells. First, miR-126 expression was analyzed using qRT-PCR in 4 human CRC cell lines (SW480, SW620, HT-29 and HCT-116). Furthermore, the biological properties of miR-126 in CRC cells in vitro were examined by applying Cell Counting Kit 8, cell cycle, cell apoptosis and transwell assays. The mechanisms and pathways of miR-126-mediated in CRC cells were detected by using qRT-PCR, western blotting and luciferase reporter assay. We found that miR-126 overexpression inhibited cell proliferation, migration and invasion, and induced cell arrest in the G0/G1 phase of CRC cells, suggesting that miR-126 functions as a tumor suppressor in CRC cells. Furthermore, we identified the CXC chemokine receptor 4 (CXCR4) as a target of miR-126, and showed that it was negatively regulated by miR-126. We demonstrated that miR-126-mediated tumor suppression might be partly dependent on AKT and ERK1/2 signaling pathways. In conclusion, our data revealed that miR-126 functions as a tumor suppressor in CRC cells by regulating CXCR4 expression via the AKT and ERK1/2 signaling pathways and might be a novel target for therapeutic strategies in CRC.