Cell division cycle 5-like protein (Cdc5L) is a core component of the putative E3 ubiquitin ligase complex containing Prp19/Pso4, Plrg1 and Spf27. This complex has been shown to have a role in pre-messenger RNA splicing from yeast to humans; however, more recent studies have described a function for this complex in the cellular response to DNA damage. Here, we show that Cdc5L interacts physically with the cell-cycle checkpoint kinase ataxia-telangiectasia and Rad3-related (ATR). Depletion of Cdc5L by RNA-mediated interference methods results in a defective S-phase cell-cycle checkpoint and cellular sensitivity in response to replication-fork blocking agents. Furthermore, we show that Cdc5L is required for the activation of downstream effectors or mediators of ATR checkpoint function such as checkpoint kinase 1 (Chk1), cell cycle checkpoint protein Rad 17 (Rad17) and Fanconi anaemia complementation group D2 protein (FancD2). In addition, we have mapped the ATR-binding region in Cdc5L and show that a deletion mutant that is unable to interact with ATR is defective in the rescue of the checkpoint deficiency in Cdc5L-depleted cells. These findings show a new function for Cdc5L in the regulation of the ATR-mediated cell-cycle checkpoint in response to genotoxic agents.