Abstract: AIM:To observe the changes of Notch signaling pathway related molecules after hypoxia/reoxygenation and the effect of Notch signaling pathway blockage on apoptosis induced by myocardial hypoxia/reoxygenation. METHODS: Cardiac myocytes were divided into normoxic group and hypoxia/reoxygenation group and each group was further divided into three groups: control group, DMSO group and GSI (gamma secretase inhibitors) group. Notch signaling molecule mRNA and protein expression level were detected by real-time PCR and Western blot. Apoptotic cells were observed by TUNEL staining and intracellular ROS levels were assayed using fluorescence probe DCFH-DA. RESULTS: mRNA and protein level of Notch signaling pathway related molecules and apoptosis and ROS levels increased significantly after hypoxia/reoxygenation. The administration of GSI produced no significant effects on ligand and receptor of the upstream of Notch signaling pathway but significantly inhibited the expression level of Hes1, a downstream transcription factor of Notch signaling pathway. At the same time, ROS and apoptosis further increased. CONCLUSION: Upregulation of the Notch signaling pathway after hypoxia/reoxygenation may exert protective effects on cardaic myocytes, which is probably related to the decreased level of ROS.