Significance Human dietary exposures to foods contaminated with fungi that synthesize aflatoxins present major health concerns and are associated with increased risk of hepatocellular carcinomas. Previous biochemical analyses revealed a prominent role for DNA polymerase ζ in replication bypass and mutagenesis of the long-lived aflatoxin DNA adduct. This investigation reveals an essential role for polymerase ζ in cell survival after aflatoxin exposure and that polymerase ζ limits aflatoxin-induced genomic instability and associated nuclear defects.