Abstract: Objective To investigate the regulatory effect of EZH2 on the FAK/F-actin/ROS pathway, and analyze its effects on the proliferation, invasion, and migration of colorectal cancer cells. Methods Twenty-four subcutaneous transplanted tumor models of nude mice were divided into the EZH2 NC group, EZH2 mimic group, EZH2 NC+ cytochalasin D group, and EZH2 mimic+ cytochalasin D group with 6 mice in each group. The tumor growth was observed after 14 days of incubation. After culture of human CRC cell lines SW480 and SW620 cells, SW480 cells were divided into 4 groups of a, b, c, and d by liposome transfection method: EZH2 NC was group a, EZH2 mimic was group b, EZH2 NC added cytochalasin D intervention was group c, and EZH2 mimic added cytochalasin D intervention was group d. SW620 cells were divided into groups e, f, g, and h, and the cell grouping treatment was the same as that of SW480 cells. Western blot assay was used to detect the expression of FAK/F-actin/ROS pathway-related proteins in SW480 cells of each group, and immunofluorescence staining was used to observe the expression and distribution of F-actin. Cell scratch assay was used to detect the migration ability of two kinds of cells, and Transwell migration assay was used to detect the migration ability of two kinds of cells. CCK-8 assay was used to detect the activity of the two cells. Results The tumor volume of the EZH2 mimic group was significantly larger than that of the EZH2 NC group (P<0.05), and the tumor volume intervened by cytochalasin D was significantly reduced compared with that before (P<0.05), and there was no significant difference between the two groups (P>0.05). The protein expression levels of EZH2, p-FAK, p-Paxillin, NOX2, NOX4, and p-JNK in the EZH2 mimic group were significantly higher than those in the EZH2 NC group (P<0.05), and the protein expression level of RUNX3 was slightly lower than that in the EZH2 NC group (P<0.05). The number of F-actin distributions, migration capacity, and cell viability were increased. After cytochalasin D intervention, the protein expression levels of EZH2, p-FAK, and p-Paxillin were not significantly different from before (P>0.05), the protein expression levels of NOX2, NOX4, and p-JNK were significantly lower than before (P<0.05), and the protein expression level of RUNX3 was significantly higher than before (P<0.05). There was no significant difference between the two groups (P>0.05), and the F-actin distribution number, migration ability, and cell viability were reduced, with no significant difference between the two groups (P>0.05). Conclusion EZH2 can promote the proliferation, invasion and metastasis of colorectal cancer cells by up-regulating FAK/F-actin/ROS pathway activity.