The acetylation of histone H3 on lysine 56 (H3-K56) occurs during S phase and contributes to the processes of DNA damage repair and histone gene transcription. Hst3 and Hst4 have been implicated in the removal of histone H3-K56 acetylation in Saccharomyces cerevisiae. Here, we show that Hst3 and Hst4 regulate the replicative lifespan of S. cerevisiae mother cells. An hst3Δ hst4Δ double-mutant strain, in which acetylation of histone H3-K56 persists throughout the genome during the cell cycle, exhibits genomic instability, which is manifested by a loss of heterozygosity with cell aging. Furthermore, we show that in the absence of other proteins Hst3 and Hst4 can deacetylate nucleosomal histone H3-K56 in a nicotinamide adenine dinucleotide(NAD)+-dependent manner. Our results suggest that Hst3 and Hst4 regulate replicative lifespan through their ability to deacetylate histone H3-K56 to minimize genomic instability.