Aim To study the effect of Notch1/Hes1 pathway activation on the cardiomyocyte hypertrophy induced by high glucose (HG), and observe its role in oxidative stress. Methods Primarily cultured rat cardiomyocytes were used in the study. The cell surface areas of the cardiomyocytes were measured by an image analysis system. The cell protein content was detected by BCA method. The expression levels of Notch1, Hes1, superoxide dismutase 1(SOD1) and induction nitric oxide synthase(iNOS) were determined by RT-qPCR and Western blot. Related kits were used to measure the content of malondialdehyde(MDA) and mtric oxide(NO) in cardiomyocytes. Results Compared with control group, the cell surface areas and total protein content of the cardiomyocytes were significantly increased (P<0.05), the expression levels of Notch1 and Hes1 were significantly decreased (P<0.05), the levels of MDA, NO and iNOS were significantly increased (P<0.05), and the expression of SOD1 was greatly decreased (P<0.05) in HG-induced cardiomyocytes. Notch1 activation suppressed HG-induced cardiomyocyte hypertrophy and the levels of oxidative stress (P<0.05). These effects of Notch1 activation were abolished by Notch1 gene interference (P<0.05). Conclusion Notch1 activation reduces HG-induced cardiomyocyte hypertrophy by decreasing oxidative stress in cardiomyocyte.