© 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd In eukaryotic cells, a surveillance mechanism, the S phase checkpoint, detects and responds to insults that challenge chromosomal replication, arresting cell cycle progression and triggering appropriate events to prevent genomic instability. In the budding yeast Saccharomyces cerevisiae, Mec1/ATM/ATR, and its downstream kinase, Rad53/Chk2, mediate the response to genotoxic stress. In this study, we place Cip1, a recently identified Cdk1 inhibitor (CKI), under the regulation of Mec1 and Rad53 in response to genotoxic stress. Cip1 accumulates dramatically in a Mec1- and Rad53-dependent manner upon replication stress. This increase requires the activity of MBF, but not the transcriptional activator kinase Dun1. At the protein level, stabilization of replication stress-induced Cip1 requires continued de novo protein synthesis. In addition, Cip1 is phosphorylated at an S/TQ motif in a Mec1-dependent manner. Deletion of Cip1 affects proliferation in hydroxyurea-containing plates. Significantly, the sensitivity is increased when the dosage of the G1 cyclin CLN2 is increased, compatible to a role of Cip1 as a G1-cyclin-dependent kinase inhibitor. In all, our results place Cip1 under the S phase checkpoint response to genotoxic stress. Furthermore, Cip1 plays a significant role to preserve viability in response to insults that threaten chromosome replication.