Abstract: AIM To observe the effect of stimulation of the Notch signaling pathway on apoptosis induced by myocardial hypoxia/reoxygenation (H/R). METHODS Cardiac myocytes were divided into four groups:Normoxia+OP9-GFP group (OP9, stable cell line; GFP, green fluorescent protein), Normoxia+OP9-Dll1 group (Dll1, Delta-like 1, Notch ligand), H/R+OP9-GFP group and H/R+OP9-Dll1 group. Apoptotic cells were observed by flow cytometry and intracellular reactive oxygen species (ROS) levels were assayed by fluorescence probe DCFH-DA. RESULTS The apoptotic cells significantly increased after hypoxia/reoxygenation (P<0.01). However, the apoptotic cells decreased when the cardiac myocytes were co-cultured with OP9-Dll1 (P<0.01), which produced Notch ligands. Intracellular ROS levels, which were significantly increased after hypoxia/reoxygenation (P<0.01), were reduced by co-cultured OP9-Dll (P<0.01). Paraquat used to increase intracellular ROS significantly inhibited anti-apoptotic effects of the Notch pathway after hypoxia/reoxygenation (P<0.01). CONCLUSION Activation of the Notch pathway after hypoxia/reoxygenation elicits anti-apoptotic effects on cardaic myocytes, which is related to decreased intracellular ROS levels.