Distinct cellular DNA damage repair pathways maintain the structural integrity of DNA and protect it from the mutagenic effects of genotoxic exposures and processes. The occurrence of O-6-carboxymethylguanine (O-6-CMG) has been linked to meat consumption and hypothesized to contribute to the development of colorectal cancer. However, the cellular fate of O-6-CMG is poorly characterized and there is contradictory data in the literature as to how repair pathways may protect cells from O-6-CMG mutagenicity. To better address how cells detect and remove O-6-CMG, we evaluated the role of two DNA repair pathways in counteracting the accumulation and toxic effects of O-6-CMG. We found that cells deficient in either the direct repair protein O-6-methylguanine-DNA methyltransferase (MGMT), or key components of the nucleotide excision repair (NER) pathway, accumulate higher levels O-6-CMG DNA adducts than wild type cells. Furthermore, repair-deficient cells were more sensitive to carboxymethylating agents and displayed an increased mutation rate. These findings suggest that a combination of direct repair and NER circumvent the effects O-6-CMG DNA damage.