The packaging of eukaryotic DNA into chromatin is likely to be crucial for the maintenance of genomic integrity. Histone acetylation and deacetylation, which alter chromatin accessibility, have been implicated in DNA damage tolerance. Here we show that Schizosaccharomyces pombe Hst4, a homolog of histone deacetylase Sir2, participates in S-phase-specific DNA damage tolerance. Hst4 was essential for the survival of cells exposed to the genotoxic agent methyl methanesulfonate (MMS) as well as for cells lacking components of the DNA damage checkpoint pathway. It was required for the deacetylation of histone H3 core domain residue lysine 56, since a strain with a point mutation of its catalytic domain was unable to deacetylate this residue in vivo. Hst4 regulated the acetylation of H3 K56 and was itself cell cycle regulated. We also show that MMS treatment resulted in increased acetylation of histone H3 lysine 56 in wild-type cells and hst4Delta mutants had constitutively elevated levels of histone H3 K56 acetylation. Interestingly, the level of expression of Hst4 decreased upon MMS treatment, suggesting that the cell regulates access to the site of DNA damage by changing the level of this protein. Furthermore, we find that the phenotypes of both K56Q and K56R mutants of histone H3 were similar to those of hst4Delta mutants, suggesting that proper regulation of histone acetylation is important for DNA integrity. We propose that Hst4 is a deacetylase involved in the restoration of chromatin structure following the S phase of cell cycle and DNA damage response.