〔Abstract〕 Objective To analyze the effect of procaine(PCA) on the methylation status and expression level of Septin9, and to explore the molecular mechanism of PCA inhibiting the proliferation of colon cancer DLD-1 and HCT116 cells. Methods Different concentrations of PCA treated colon cancer cells at different times, and MTS method was used to detect cell proliferation activity. Flow cytometry and Transwell experiments were used to detect the effects of PCA on cell cycle, apoptosis and migration ability; BGS method and qRT-PCR were used to detect the methylation status and expression level of Septin9 before and after PCA treatment. Results PCA inhibited the proliferation of DLD-1 and HCT116 cells in a concentration-and time-dependent manner(P<0.05). Compared with the control group, PCA treatment blocked cell growth in G2/M phase, significantly induced apoptosis and inhibited cell migration(P<0.05). After PCA treatment, the degree of Septin9 methylation decreased, the expression of Septin9 mRNA increased, and the differences were statistically significant(P<0.05). Conclusion PCA can inhibit the proliferation and migration of colon cancer cells by reversing Septin9 methylation.