Hepatitis C virus (HCV) NS3 protein possesses protease and helicase activities and is considered an oncoprotein in virus-derived hepatocellular carcinoma. The NS3-associated oncogenesis has been studied but not fully understood. In this study, we have identified novel interactions of the NS3 protein with DNA repair factors, Werner syndrome protein (WRN) and Ku70, in both an HCV subgenomic replicon system and Huh7 cells expressing NS3. HCV NS3 protein inhibits WRN-mediated DNA repair and reduces the repair efficiency of nonhomologous end joining. It interferes with Ku70 recruitment to the double-strand break sites and alters the nuclear distribution of WRN-Ku repair complex. In addition, WRN is a substrate of the NS3/4A protease; the level of WRN protein is regulated by both the proteasome degradation pathway and HCV NS3/4A protease activity. The dual role of HCV NS3 and NS3/4A proteins in regulating the function and expression level of the WRN protein intensifies the effect of impairment on DNA repair. This may lead to an accumulation of DNA mutations and genome instability and, eventually, tumor development. IMPORTANCE HCV infection is a worldwide problem of public health and a major contributor to hepatocellular carcinoma. The single-stranded RNA virus with RNA-dependent RNA polymerase experiences a high error rate and develops strategies to escape the immune system and hepatocarcinogenesis. Studies have revealed the involvement of HCV proteins in the impairment of DNA repair. The present study aimed to further elucidate mechanisms by which the viral NS3 protein impairs the repair of DNA damage. Our results clearly indicate that HCV NS3/4A protease targets WRN for degradation, and, at the same time, diminishes the repair efficiency of nonhomologous end joining by interfering with the recruitment of Ku protein to the DNA double-strand break sites. The study describes a novel mechanism by which the NS3 protein influences DNA repair and provides new insight into the molecular mechanism of HCV pathogenesis.