Central to the replication checkpoint are two protein kinases, ATR, and its downstream target kinase, Chk1. Signaling pathways leading to activation of ATR-Chk1 have been extensively investigated; however, events that mediate checkpoint termination and replication fork restart are less well understood. Here, we define a coupled activation-destruction mechanism of Chk1 that regulates checkpoint termination and cellular sensitivity to replicative stress. DNA damage-induced phosphorylation or mutation of a conserved motif of Chk1 both activates Chk1 and exposes a degron-like region at the carboxyl-terminus of Chk1 to a Fbx6-containing SCF (Skp1-Cul1-F-box) E3 ligase, which mediates the ubiquitination and degradation of Chk1, and, in turn, terminates the checkpoint. The expression levels of Chk1 and Fbx6 proteins showed an inverse correlation in both cultured cancer cell lines and in a small cohort of human breast tumor tissues. Further, we show that low levels of Fbx6 and consequent impairment of replication stress-induced Chk1 degradation are associated with cancer cell resistance to killing by the chemotherapeutic agent, camptothecin (CPT). We propose that Fbx6-dependent Chk1 degradation contributes to S-phase checkpoint termination, and that a defect in this mechanism might increase tumor cell resistance to certain anticancer drugs.