Eco1/Eso1 protein plays an important role in chromosome segregation, DNA repair and gene regulation. Eco1 mutation induces Roberts syndrome clinically and rDNA transcription disorders in vivo. In this study, we examined the role of Eso1 protein binding to polymerase 5 (Pol5) and the acetylation of Pol5 protein in the regulation of Schizosaccharomyces pombe (S. pombe) viability. Immunoprecipitation and mass spectrometry assays identified Eso1 protein binding to Cdc2, Pol5 and Cdc21, as well as other proteins. Pol5 protein specifically bound to Eso1 protein, but not to the Rad30 part or Rad30 part plus the additional zinc finger domain of Eco1 protein. Mass spectrometry data further identified several acetylation or trimethylation modification sites in the lysine residues of the Pol5 protein. However, the mutation of the Pol5 K47 site to arginine was lethal to S. pombe. Eso1 protein was able to acetylate Pol5 protein and mediate S. pombe viability. On the whole, our data indicate that the Eso1 interaction with Pol5 which acetylates Pol5 protein is essential for S. pombe viability.