Objective To investigate the effects of USP7 on the stemness characteristics and oxaliplatin resistance of human colon cancer HCT116 cells. Methods Oxaliplatin-resistant HCT116 cell line was established, and the expression level of USP7 was detected. Si-CON, USP7-si1, and USP7-si2 were transfected respectively, and the knockdown efficiency was verified by immunoblotting. Colony formation assays were used to assess cell proliferation ability. qPCR was performed to detect the expression levels of cancer stemness-related genes. CCK-8 assays were employed to evaluate the sensitivity of cells to oxaliplatin. Results USP7 expression was upregulated in oxaliplatin-resistant HCT116 cells. Knockdown of USP7 significantly inhibited the proliferation ability of HCT116 cells (P<0.01) and suppressed the expression of cancer stemness-related genes and oxaliplatin resistance (P<0.01). Conclusion USP7 can promote the stemness characteristics of colon cancer HCT116 cells and mediate oxaliplatin resistance.