A previous study demonstrated that miR-126 expression was significantly downregulated in highly metastatic colon cancer cells. This study was to investigate the biological function of miR-126 and its regulation of target genes in colon cancer cells. Quantitative PCR was used to detect miR-126 expression in colon cancer SW480 and SW620 cells. MTT assay was to measure the changed cell viability after miR-126 mimics transfection. Wound healing and Transwell migration and invasion assays measured capacity of tumor cell migration and invasion of SW480 and SW620 cells after miR-126 transfection. Luciferase reporter assay and Western blot were used to assess both transcriptional and expression levels of one of the miR-126 target genes (i.e., CXCR4). Levels of miR-126 expression were lower in colon cancer SW480 and SW620 cells than in the adjacent normal epithelial tissues (P < 0.05). Transfection of miR-126 mimics significantly reduced colon cancer cell viability compared to NC cells (P < 0.05). The wound healing and Transwell migration and invasion assays showed that miR-126 mimics inhibited SW480 and SW620 cell migration and invasion capacity. Bioinformatics predicted that CXCR4 is one of the miR-126 target genes. Indeed, luciferase reporter assay and Western blot confirmed that CXCR4 is a miR-126 target gene. Expression of miR-126 inhibited colon cancer cell viability and reduced tumor cell migration and invasion capacity by its negative regulation of CXCR4 expression.