Objective To investigate the effect of over-expression of nudix hydrolase 21 (NUDT21) on the proliferation of colon cancer HCT-116 cells and its mechanism. Methods The NUDT21 over-expression plasmid was constructed by Gibson assembly. The colony formation assay and CCK-8 assay were used to detect cell proliferation. The cell cycle of HCT-116 cells was detected by flow cytometry. Western blot was performed to detect the expressions of P53, cyclin-dependent kinase 2 (CDK2), phosphorylated retinoblastoma protein at serine 780 (p-Rb-Ser780), and p-Rb-Ser608. Results The sequencing results showed that the NUDT21 over-expression plasmid was successfully constructed. After the NUDT21 over-expression plasmid was transfected into HCT-116 cells, the expressions of NUDT21 mRNA and protein in the cells were significantly increased. The over-expression of NUDT21 inhibited the proliferation of HCT-116 cells and arrested the cell cycle in G0/G1 phase. The expressions of CDK2, p-Rb-Ser608, and p-Rb-Ser780 proteins decreased while the expression of P53 protein increased. Conclusion Over-expression of NUDT21 inhibits the proliferation of HCT-116 cells by blocking P53/CDK2/Rb signal pathway.