Rad9 is required for the activity of the genotoxin-induced checkpoint signaling pathway to control cell cycle progress and maintain genomic stability. In the fission yeast S. Pombe and chicken cells, the Rad9 gene is essential for prevention of premature chromosomal condensation (PCC:S/M checkpoint control). However, precise features in the S/M checkpoint controlled by Rad9 in mammalian cells are still not clear. In this study, mouse embryonic stem (ES) cells with a targeted deletion of Mrad9, the counterpart of S. pombe rad9, were used to evaluate the detailed function of Mrad9 in S/M checkpoint control. We found that Mrad9 deletion from ES cells led to failure of S/M checkpoint control across the entire S phase, and was alleviated by introducing mouse or human Rad9 into the Mrad9-deleted cells. We also found that the phosphorylation sites on Tyr28 and the C-terminus of Rad9 are required for this checkpoint control. Moreover, the DNA replication inhibitor hydroxyurea (HU) induced cdc2 Tyr15 phosphorylation and increased 14-3-3sigma protein levels in Mrad9(+)/(+) ES cells, but failed to do so in Mrad9(-)/(-) ES cells. Taken together, these results suggest that phosphorylation of Rad9 plays a critical role in the activation of the S/M checkpoint, and that downstream proteins cdc2 and 14-3-3sigma mediate this function.