The Chk1 protein is essential for genome integrity maintenance and cell survival in eukaryotic cells. After prolonged replication stress, Chk1 can be targeted for proteasomal degradation to terminate checkpoint signaling after DNA repair finishes. To ensure proper activation of DNA damage checkpoint and DNA repair signaling, a steady-state level of Chk1 needs to be retained under physiological conditions. Here, we report a dynamic signaling pathway that tightly regulates Chk1 stability. Under unperturbed conditions and upon DNA damage, ataxin-3 (ATX3) interacts with Chk1 and protects it from DDB1/CUL4A- and FBXO6/CUL1-mediated polyubiquitination and subsequent degradation, thereby promoting DNA repair and checkpoint signaling. Under prolonged replication stress, ATX3 dissociates from Chk1, concomitant with a stronger binding between Chk1 and its E3 ligase, which causes Chk1 proteasomal degradation. ATX3 deficiency results in pronounced reduction of Chk1 abundance, compromised DNA damage response, G2/M checkpoint defect and decreased cell survival after replication stress, which can all be rescued by ectopic expression of ATX3. Taken together, these findings reveal ATX3 to be a novel deubiquitinase of Chk1, providing a new mechanism of Chk1 stabilization in genome integrity maintenance.