Objective In order to investigate the effects of acylglycerol kinase(AGK) on proliferation and migration in HCT116 cells. Methods In this study, shRNA silencing lentivirus plasmid of AGK gene was constructed, and the second generation lentivirus packaging system was used to prepare and concentrate lentivirus venom and infect human colon cancer cell HCT⁃116 cell line.Then HCT⁃116 cell lines with knockdown AGK gene were screened for puromycin resistance.qRT⁃PCR and Western blotting were used to detect the mRNA and protein expression of AGK after knockdown. CCK⁃8 kit was used to detect the proliferation of AGK knockdown stable cell lines.The effect of AGK knockout on cell migration was detected by cells scratch assay. Results ShRNA silencing lentivirus plasmid of AGK gene constructed in this experiment can effectively knockdown endogenous AGK in HCT116.The mRNA and protein levels of AGK in HCT116⁃ASR⁃876 and HCT116⁃ASR⁃878 cells were significantly lower than those of wild⁃type cell lines (P<0.001).CCK⁃8 detection showed that AGK knockdown significantly reduced cell proliferation ability (P<0.05).The scratch test showed that AGK knockdown could significantly inhibit cell migration. Conclusion Knockout of AGK gene can significantly reduce the proliferation and migration abilities of HCT116 cells, which might provide a new potential target for targeted therapy of colorectal cancer.