Background Propofol, an extensively used intravenous anesthetic agents during cancer resection surgery, has been confirmed to execute anti-tumor effect on multiple cancers, including colorectal cancer (CRC). Although the role of propofol in CRC has been previously reported, its action mechanism remains poorly understood. This study further explored the biological function and underlying mechanism of propofol in CRC cells. Methods The cell proliferation, migration and invasion were assessed by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, wound healing assay and transwell assay, respectively. The expression levels microRNA-124-3p.1 (miR-124-3p.1) and AKT serine/threonine kinase 3 (AKT3) was analyzed by quantitative real-time polymerase chain reaction. Western blot assay was employed to measure the protein expression of MMP-9, Vimentin and Cyclin D1. The interaction between miR-124-3p.1 and AKT3 was predicted by TargetScan and confirmed by dual-luciferase reporter assay. Results Propofol inhibited CRC cell proliferation, migration and invasion. Knockdown of miR-124-3p.1 or AKT3 upregulation reversed the inhibitory effects of propofol on CRC cell proliferation and metastasis. Besides, AKT3 was a direct target of miR-124-3p.1 and its overexpression abated the anti-tumor effect of miR-124-3p.1 on CRC cell proliferation and metastasis. Conclusion Propofol inhibited CRC cell proliferation, migration and invasion by upregulating miR-124-3p.1 and downregulating AKT3, providing a new sight for propofol treatment of CRC.