Objective: To explore the expression of microRNA-340 and rho associated-coiledcoil forming protein kinase (ROCK1) in tissues of colon cancer and its clinical significance. Methods: A total of 41 tissue specimens of colon cancer and adjacent normal tissues were chosen clinically. Quantitative real-time PCR were used to detect the different expression of miR-340 and ROCK1 mRNA in tissues. Western bloting assay was used to detect the expression of RohA/ROCK pathway. Results: miR-340 mRNA decreased significantly in colon cancer tissue (0.757±0.056) compared with the adjacent normal tissue (1.290±0.082), and the difference had statistical significance (P<0.05). ROCK1 mRNA increased significantly in colon cancer tissue (1.551±0.078) compared with the adjacent normal tissue (0.823±0.059), and the difference had statistical significance (P<0.05). The expression levels of ROCK1, ROCK2 and RhoA in colorectal cancer tissues were 0.455±0.038, 0.385±0.033 and 0.852±0.075, and the normal mucosal tissues were 0.185±0.021, 0.102±0.011 and 0.401±0.044, respectively. Those in the cancer tissues were higher than normal tissues, with statistically significant differences (P<0.05). The expression levels of miR-340 and ROCK1 were not associated with gender, age, locations, depth of tumor invasion and tumor differentiation (P>0.05). However, the expression levels of miR-340 and ROCK1 were associated with TNM stage and lymph node metastasis (P<0.05). Pearson correlation analysis revealed that the expression of miR-340 had a close negative correlation with ROCK1 in colon cancer (r=−0.509, P<0.001). Conclusion: The low expression of miR-340 and high expression of ROCK1 are closed associated with TNM stage and lymph node metastasis. miR-340 may act as oncogene via targeting ROCK1 in colon cancer.