Objective: To investigate the effect of microRNA-139-5p (miR-139-5p) expression in colorectal cancer and its influence on migration and invasion ability of colorectal cancer cells. Methods: The expression alteration of miR-139-5p in colorectal cancer tissue and different colorectal cancer cell lines were detected by using fluorescent quantitative PCR. The influence of miR-139-5p transfection or miR-139-5p inhibitor treatment on migration and invasion ability of colorectal cancer cells were detected by Boyden chamber assay and wound healing assay. The target gene of miR-139-5p was predicted by bioinformatics analysis and was identified by luciferase reporter assay, and then the influence of miR-139-5p transfection on its target gene expression was determined by Western blot analysis. Results: The miR-139-5p mRNA expressions in both colorectal cancer tissue and colorectal cancer cell lines were significantly decreased compared with corresponding control (all P<0.05). The migration and invasion ability in colorectal cancer DLD1 and HCT116 cells were significantly decreased after miR-139-5p transfection and were significantly increased after miR-139-5p inhibitor treatment (all P<0.05). Bioinformatics analysis showed that Notch1 was the potential target gene of miR-139-5p which was then identified by luciferase reporter assay. Western blot results showed that Notch1 protein expressions in DLD1 and HCT116 cells were significantly down-regulated after miR-139-5p transfection (both P<0.05). Conclusion: MiR-139-5p may inhibit the migration and invasion of cancer cells through regulating its target gene Notch1, so the down-regulated miR-139-5p may play an important role in the occurrence and development colorectal cancer.