Acetaldehyde, a chemical that can cause DNA damage and contribute to cancer, is prevalently present in our environment, e.g. in alcohol, tobacco, and food. Although aldehyde potentially promotes crosslinking reactions among biological substances including DNA, RNA, and protein, it remains unclear what types of DNA damage are caused by acetaldehyde and how they are repaired. In this study, we explored mechanisms involved in the repair of acetaldehyde-induced DNA damage by examining the cellular sensitivity to acetaldehyde in the collection of human TK6 mutant deficient in each genome maintenance system. Among the mutants, mismatch repair mutants did not show hypersensitivity to acetaldehyde, while mutants deficient in base and nucleotide excision repair pathways or homologous recombination (HR) exhibited higher sensitivity to acetaldehyde than did wild-type cells. We found that acetaldehyde-induced RAD51 foci representing HR intermediates were prolonged in HR-deficient cells. These results indicate a pivotal role of HR in the repair of acetaldehyde-induced DNA damage. These results suggest that acetaldehyde causes complex DNA damages that require various types of repair pathways. Mutants deficient in the removal of protein adducts from DNA ends such as TDP1(-/-) and TDP2(-/-) cells exhibited hypersensitivity to acetaldehyde. Strikingly, the double mutant deficient in both TDP1 and RAD54 showed similar sensitivity to each single mutant. This epistatic relationship between TDP1(-/-) and RAD54(-/-) suggests that the protein-DNA adducts generated by acetaldehyde need to be removed for efficient repair by HR. Our study would help understand the molecular mechanism of the genotoxic and mutagenic effects of acetaldehyde.