We previously reported that the E6 oncoprotein of high-risk human papillomavirus (HPV) caused genetic instability and oncogenesis by disrupting cellular DNA repair. Here, to investigate the effect of different domains of E6 on DNA double-strand break (DSB) repair, we infected normal human oral fibroblasts (NHOF) with retroviruses expressing wild-type (wt) or mutant (mt) HPV-16 E6 and examined the cellular DNA end-joining (EJ) activity. The cells expressing E6 showed not only a diminution of error-free DNA EJ but also an increase in erroneous DNA EJ capacity if compared with cells without wt E6. Analysis of DNA EJ activities from the cells expressing mt HPV-16 E6 indicated that binding to p53 and the presence of both intact zinc finger domains of E6 are necessary for inducing the E6-mediated aberrant DNA EJ activity. Also, deletion of the PDZ binding C-terminal region reduced this activity by 50%. These findings suggest that E6 can disrupt the fidelity of DSB repair via both p53-dependent and -independent pathways and the impaired fidelity might contribute to the development of genetic instability found in HPV-associated cancer.