Renal cell carcinoma (RCC) is a heterogeneous disease with resistance to systemic chemotherapy. Elevated expression of multiple drug resistance (MDR) has been suggested to be one of the mechanisms for this resistance. Here, we provide an alternative mechanism to explain RCC's resistance to chemotherapy-induced apoptosis. Never-in mitosis A-related protein kinase 1 (Nek1) plays an important role in DNA damage response and proper checkpoint activation. The association of Nek1 with the voltage-dependent anion channel (VDAC1) is a critical determinant of cell survival following DNA-damaging treatment. We report here that Nek1 is highly expressed in RCC tumor and cultured RCC cells compared to that of normal renal tubular epithelial cells (RTE). The association between Nek1 and VDAC1 is genotoxic dependent: prolonged Nek1/VDAC1 dissociation will lead to VDAC1 dephosphorylation and initiate apoptosis. Down-regulation of Nek1 expression in RCC cells enhanced their sensitivity to DNA-damaging treatment. Collectively, these results suggest that the increased Nek1 expression in RCC cells maintain persistent VDAC1 phosphorylation, closing its channel and preventing the onset of apoptosis under genotoxic insults. Based on these results, we believe that Nek1 can serve as a potential therapeutic target for drug development in the treatment of RCC.