3-Nitrobenzanthrone (3-NBA), a potent mutagen and suspected human carcinogen, is a common environmental pollutant. Genotoxicity of 3-NBA has been associated with its ability to form the DNA adducts, including N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (C8-dG-ABA). To investigate the molecular mechanism of C8-dG-ABA mutagenesis in human cells, we have replicated a plasmid containing a single C8-dG-ABA in human embryonic kidney (HEK) 293T cells, which yielded 14% mutant progeny. The major types of mutations induced by C8-dG-ABA were G→T > G→A > G→C. siRNA knockdown of the translesion synthesis (TLS) DNA polymerases (pols) in HEK293T cells indicated that pol η, pol κ, pol ι, pol ζ, and Rev1 each has a role in replication across this adduct. The extent of TLS was reduced with each pol knockdown, but the largest decrease (of ~55% reduction) in TLS occurred in cells with knockdown of pol ζ. Pol η and pol κ were considered the major contributors of the mutagenic TLS, since mutation frequency (MF) dropped by 70%, when these pols were simultaneously knocked down. Rev1 also is important for mutagenesis, as reflected by 60% reduction in MF upon Rev1 knockdown, but it probably plays a non-catalytic role by physically interacting with the other two Y-family pols. In contrast, pol ζ appeared to be involved in the error-free bypass of the lesion, since MF increased by 60% in pol ζ knockdown cells. These results provide important mechanistic insight of the bypass of the C8-dG-ABA adduct.