We report evidence suggesting that methyltransferase SET8 plays a novel role in regulating cell cycle by suppressing DNA replication through histone binding. First, the distribution of SET8 is strongly cell cycle-dependent. SET8 was concentrated in the nucleus during G(1) and G(2) phases, and was excluded from the nucleus during S phase. Second, at G(1)/S transition, SET8 was degraded through ubiquitination via SCF/Skp2. Third, it was evident that the SET8 binds to the H4 N-terminal tail (H4NT) and blocks the acetylation of lysine residues K5, K8 and K12 of histone H4 during G(1). Such a blockage can hinder DNA replication. Fourth, SET8 binds to hypoacetylated but not hyperacetylated H4NT. Finally, overexpressing the histone-binding domain of SET8 appeared to suppress DNA replication and arrest the cell cycle before the G(1)/S transition. Taken together, these findings suggest that SET8 can be a negative regulator of DNA replication and the destruction of SET8 is required for the onset of S phase.