Chemotherapy resistance plays a pivotal role in the prognosis and therapeutic failure of patients with colorectal cancer (CRC). Cisplatin-resistant cells exhibit an inherent ability to evade the toxic chemotherapeutic drug effects which are characterized by the activation of slow-cycle programs and DNA repair. Among the elements that lead to cisplatin resistance, O6-methylguanine (O6-MG)-DNA-methyltransferase (MGMT), a DNA-repair enzyme, performs a quintessential role. In this study, we clarify the significant involvement of MGMT in conferring cisplatin resistance in CRC, elucidating the underlying mechanism of the regulatory actions of MGMT. A notable upregulation of MGMT in cisplatin-resistant cancer cells was found in our study, and MGMT repression amplifies the sensitivity of these cells to cisplatin treatment in vitro and in vivo. Conversely, in cancer cells, MGMT overexpression abolishes their sensitivity to cisplatin treatment. Mechanistically, the interaction between MGMT and CDK1 inducing slow-cycling cells is attainted via the promotion of ubiquitination degradation of CDK1. Meanwhile, to achieve nonhomologous end joining, MGMT interacts with XRCC6 to resist chemotherapy drugs. Our transcriptome data from samples of 88 patients with CRC suggest that MGMT expression is co-related with the Wnt signaling pathway activation, and several Wnt inhibitors can repress drug-resistant cells. In summary, our results point out that MGMT is a potential therapeutic target and predictive marker of chemoresistance in CRC.