Globally, gastric cancer is the second leading cause of cancer deaths because of the lack of effective treatments for patients with advanced tumors when curative surgery is not possible. Thus, there is an urgent need to identify molecular targets in gastric cancer that can be used for developing novel therapies and prolonging patient survival. Checkpoint kinase 1 (Chk1) is a crucial regulator of cell cycle transition in DNA damage response (DDR). In our study, we report that Chk1 plays an important role in promoting gastric cancer cell survival and growth, which serves as an effective therapeutic target in gastric cancer. First, Chk1 ablation by small interfering RNA could significantly inhibit cell proliferation and sensitize the effects of ionizing radiation (IR) treatment in both p53 wild type gastric cancer cell line AGS, and p53 mutant cell line MKN1. Secondly, we tested the anticancer effects of Chk1 chemical inhibitor LY2606368, which is a novel Chk1/2 targeted drug undergoing clinical trials in many malignant diseases. We found that LY2606368 can induce DNA damage, and remarkably suppress cancer proliferation and induce apoptosis in AGS and MKN1 cells. Moreover, we identified that LY2606368 can significantly inhibit homologous recombination (HR) mediated DNA repair and thus showed marked synergistic anticancer effect in combination with poly (ADP- ribose) polymerase 1 (PARP1) inhibitor BMN673 in both in vitro studies and in vivo experiments using a gastric cancer PDx model. The synergy between LY2606368 and PARP1 was likely caused by impaired the G2M checkpoint due to LY2606368 treatment, which forced mitotic entry and cell death in the presence of BMN673. In conclusion, we propose that Chk1 is a valued target for gastric cancer treatment, especially Chk1 inhibitor combined with PARP inhibitor may be a more effective therapeutic strategy in gastric cancer.