Chromatin endowed by histone modifications governs chromatin structure, which in turn represents a means to regulate cellular processes, including transcription and heterochromatin formation. Recent evidence revealed a plethora of enzymes that catalyze specific histone modifications for epigenetic maintenance, and dysregulation of which contributes to tumorigenesis and developmental defects. The histone methyltransferase SET8 (also known as Pr-Set7) was previously reported to monomethylate Lys(20) of histone H4. However, the temporal and spatial control of SET8 activity remains elusive. Here, we provide evidence to support that SET8 monomethylates Lys(20) of histone H4 during S phase by tethering to proliferating cell nuclear antigen via a putative proliferating cell nuclear antigen-interacting protein box. In addition, we show that SET8 function is required for S phase progression. Finally, deletion of SET8 in mice causes embryonic lethality, suggesting that SET8 plays an important role in mammalian embryogenesis.