7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) is one of the most common DNA lesions induced by oxidative stress. This lesion can be bypassed by DNA polymerase eta (Pol eta) using in vitro translesion synthesis (TLS) reactions. However, the role that Pol eta plays in vivo contributing to 8-oxo-dG mutagenesis remains unclear. To clarify the role of Pol eta in 8-oxo-dG mutagenesis, we have used an siRNA knockdown approach in combination with a supF shuttle vector (pSP189) which replicates in mammalian cells. The pSP189 plasmid was treated with methylene blue plus light (MBL), which produces predominantly 8-oxo-dG in DNA, and was then replicated in GM637 cells in presence of siRNA that knocks down the expression of Pol eta, or in XP-V cells, which lack functional Pol eta. The mutant frequencies were increased in the Pol eta siRNA knockdown cells and in XP-V cells relative to control, meaning that Pol eta plays an important role in preventing 8-oxo-dG mutagenesis. In the same system, knockdown of OGG1 also led to an increase in mutagenesis. Neither the type of mutations nor their distribution along the supF gene were significantly different between control and target specific siRNA-transfected cells (or XP-V cells) and were predominantly G to T transversions. These results show that Pol eta has an important role in error-free 8-oxo-dG lesion bypass and avoidance of oxidative stress-induced mutagenesis in vivo.