Oxaliplatin (L-OHP) is a standard treatment drug for colorectal cancer (CRC), but acquired drug resistance limits the outcome of patients. We investigated the involvement of sirtuin 1 (SIRT1) in L-OHP resistance in the setting of CRC via microRNA-20b-3p/DEP domain containing 1 (miR-20b-3p/DEPDC1) axis. CRC tissues that were resistant or sensitive to L-OHP were harvested, in which SIRT1, miR-20b-3p, and DEPDC1 levels were tested. L-OHP-resistant-resistant CRC cells were transfected, subsequently, cellular proliferation, invasion, migration, and apoptosis were tested, and tumor resistance to L-OHP was observed. The binding of SIRT1 to miR-20b-3p promoter and the targeting relationship between miR-20b-3p and DEPDC1 were verified. An aberrant elevation in SIRT1 expression was seen in L-OHP-resistant CRC tissues and cells. Knockdown of SIRT1 sensitized CRC cells and xenografted CRC tumors to L-OHP. SIRT1 bound with miR-20b-3p promoter to regulate DEPDC1. Reducing miR-20b-3p or raising DEPDC1 levels weakened the effect of SIRT1 knockdown on L-OHP-resistant-CRC cells. SIRT1 enhances L-OHP resistance in CRC by mediating miR-20b-3p/DEPDC1 axis.