Abstract: AIM To investigate the role of Notch1 pathway and its underlying mechanisms in H9C2 cardiomyocytes subjected to hypoxia/reoxygenation (H/R) injury in high temperature and humidity. METHODS H9C2 cardiomyocytes were cultured and divided into six groups: control group, H/R group, high temperature and humidity group, high temperature and humidity+H/R group, high temperature and humidity+Jagged1 (an activator of Notch1)+H/R group and high temperature and humidity+vehicle+H/R group. Cell apoptosis was detected by TUNEL, mitochondrial membrane potential was assessed by JC-1 and ATP content was measured by an ATP bioluminescent assay kit. The expression of Notch1 intracellular domain (Notch1 ICD), Hairy and enhancer of split (Hes1), microtubule-associated protein1 light chain 3 (LC3) and p62 were analyzed by Western blot. RESULTS Compared with those in control group, H/R injury significantly increased cell apoptosis index (P<0.05), reduced mitochondrial membrane potential (P<0.05) and ATP content (P<0.05), and increased expressions of Notch1 ICD, Hes1 and LC3-II/I (except p62) (P<0.05). Although high temperature and humidity also increased cell apoptosis index (P<0.05), reduced mitochondrial membrane potential (P<0.05) and ATP content (P<0.05), it reduced expressions of Notch1 ICD, Hes1 and LC3-II/I (except p62) (P<0.05) compared with those in control group. In addition, compared with those in H/R group or high temperature and humidity group, cell apoptosis index further increased (P<0.05), mitochondrial membrane potential (P<0.05) and ATP content reduced (P<0.05), and the expressions of Notch1 ICD, Hes1 and LC3-II/I(except p62) decreased (P<0.05) in high temperature and humidity+H/R group. Moreover, compared with those in high temperature and humidity+H/R group, adding Jagged1 (an activator of Notch1) reduced cell apoptosis index (P<0.05) and increased the mitochondrial membrane potential (P<0.05) and ATP content (P<0.05) as well as the expressions of Notch1 ICD, Hes1 and LC3-II/I (except p62) (P<0.05). CONCLUSION Activation of Notch1 pathway can alleviate hypoxia/reoxygenation injury of H9C2 cardiomyocytes in high temperature and humidity through accelerating autophagy.