Genomic DNA that essential for cell survival is constantly undergoes various forms of DNA damages upon attacked by DNA-damaging agents from exogenous and endogenous sources. DNA-protein cross-links (DPCs) are super-bulky, steric hindrance and less characterized DNA damage among those so far identified. Currently known DPCs are classified into four main types depending on the way of attachment to DNA strands. Of these types, type 1 is the most ubiquities in which cross-linked proteins (CLPs) are covalently attached to an undistorted DNA strand. While several researchers worldwide start to be attention about DPC damage, the repair factors that are indispensable for the processing of type 1 DPC remain largely elusive. Therefore, in the present study, we analyzed the role of translesion synthesis (TLS) DNA polymerases κ and ι (polκ and ι) in the processing of type 1 DPC. Obviously, mouse cells deficient in polκ were highly sensitive to 2ʹ-deoxy-5-azacytidine (azadC, a DNA methylating agent) and formaldehyde (FA, a simple aldehyde). Furthermore, the quantitative analysis of DPCs in polκ proficient and deficient cells using fluorescence labeling method which we have developed recently revealed that the amount of DPCs increased significantly in azadC and FA-treated cells compared to untreated control. In contrast, a DNA methylation inhibitor Zebularine (Zeb) does not enhance the sensitivity of polκ deficient cells compared to polκ proficient cells. Additionally, no DPC is formed upon treatment with Zeb in polκ cells. The most remarkable conclusion is that the sensitivity of polκ deficient cells to azadC is exclusively due to DPC and ruling out the involvement of polκ in DNA methylation. Based on the current findings, we suggested a possible repair model for type 1 DPC induced by azadC and FA. Wherein, small peptides result from breakage of large CLPs are bypassed by polκ and consequently the repair proceeds.