Chk1 is an essential kinase for maintaining genome integrity and cell cycle checkpoints through phosphorylating several downstream targets. Recently, we demonstrated that Chk1 is also required for cell proliferation in somatic cells under unperturbed condition through regulating transcription of several genes. Here, we show that Chk1 is required for S phase progression and RNR2 is a critical downstream target of genes transcriptionally regulated by Chk1. Hence, although RNR2 expression reached maximum at S phase in the presence of Chk1, Chk1 depletion arrested the cell cycle at S phase and reduced RNR2 expression at both mRNA and protein levels. Ectopic expression of RNR2 failed to rescue the S phase arrest observed in Chk1 depleted cells, suggesting the presence of an additional Chk1-target(s) for completion of S phase other than RNR2. Therefore, our results suggest that Chk1 is required for DNA replication at least through regulating RNR2 gene transcription.