Background MicroRNAs (miRNAs) exerts a vital part in tumor occurrence.The role of miR-378a-5p and CDK1 in colorectal cancer (CRC) was investigated in this study. Methods The investigation of TCGA database and the detection of miR-378a-5p expression in colorectal cancer pathological tissues and colorectal cancer cell lines were performed by using qRT-PCR. We conducted cell function experiments (CCK-8, EDU, Colony formation assay, Wound healing assay, Transwell assay, Cell apoptosis detection, Cell cycle detection) and nude mouse tumor formation experiments to evaluate the effects of miR-378a-5p on proliferation, metastasis, The impact of invasion to explore the role of miR-378a-5p in vivo and in vitro. Then, through the TCGA database, immunohistochemistry of pathological tissues, and cell function experiments, the role of the target gene CDK1 of miR-378a-5p verified by database prediction and dual luciferase reporter gene experiments in colorectal cancer was explored. Finally, whether up-regulation of CDK1 restores the inhibitory effect of overexpression of miR-378a-5p on the proliferation of CRC cells is studied by overexpression of CDK1. Results Bioinformatics analysis showed significant downregulation of miR-378a-5p level within colorectal cancer (CRC). Cell function experiments and tumor xenograft mouse models confirmed the low expression of miR-378a-5p within CRC tissues, which indicated the tumor suppression role of miR-378a-5p within CRC. For better exploring the regulation of miR-378a-5p in CRC, we predicted and validated cell cycle-dependent protein kinase 1 (CDK1) to be the miR-378a-5p target geneand found that miR-378a-5p suppressed CRC cell proliferation by targeting CDK1. Conclusion To sum up, results in this work discover the mechanism of miR-378a-5p in retarding CRC cell proliferation through suppressing CDK1 expression, and it may become a possible therapeutic target to treat CRC.