Objective This study aimed to demonstrate the role of MYPT1(Myosin phosphatase-targeting subunit 1） in the development of colorectal cancer(CRC) and determine the potential mechanisms of action. Methods MRNA expression level was detected by RT-PCR, protein expression level was detected by Western blot, cell proliferation activity was detected by CCK-8 method, migration ability of colorectal cancer cells was detected by Transwell method, protein that could interact with MYPT1 was screened by bioinformatics software. The expression level of MYPT1 and its correlation with RhoA protein 1(ROCK1), A member of RAS homologous gene family, were analyzed by TCGA database. According to the experimental plan, groups were divided into blank control group, negative control group, MYPT1 overexpression group, RhoA overexpression group, MYPT1 overexpression+RhoA no-load group and MYPT1 overexpression+RhoA overexpression group.Results Compared with normal adjacent tissues and normal intestinal mucosal cells, MYPT1 was lower expressed in tissues and cells of colorectal cancer(P＜0.05). Overexpression of MYPT1 inhibited the proliferation and migration of colorectal cancer cells(P＜0.05). MYPT1 negatively regulates the expression of RhoA and ROCK1(P＜0.05). Overexpression of RhoA promotes the proliferation and migration of colorectal cancer cells(P＜0.05). This paper pressume that MYPT1 inhibited ROCK1 expression by downregulating RhoA expression(P＜0.05). MYPT1 inhibited cell proliferation and migration by negatively regulating RhoA(P＜0.05). Conclusion MYPT1 is lowly expressed in CRC cancer tissue and cells,and MYPT1 regulates the proliferative and migratory abilities via the RhoA/ROCK pathway.