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Background To investigate the effect of hydrogen peroxide (H2S) on myocardial clock gene Bmal1 in ischemic cardiomyocytes. Materials & methods Quantitative PCR (qPCR) was used to detect the expression of Bmal1 at the mRNA level in H9C2 rat cardiomyocytes. The protein expressions of Bax and Bcl-2, PI3K/Akt, caspase-3 were measured by western blotting. The levels of reactive oxygen species (ROS) were determined by ELISA. Results The expression level of clock gene Bmal1 demonstrated a clock rhythm of periodic oscillation within 24 h. Compared with the control group, H2S treatment maintained the rhythm of the clock gene in ischemic cardiomyocytes and increased the transcription and expression levels of Bmal1. H2S increased cell survival by activating PI3K/Akt signaling pathway, inhibiting mitochondrial apoptosis signaling, and reducing intracellular oxidative stress. PI3K/Akt and Bmal1 were demonstrated to be involved in H2S protection of cardiomyocyte ischemia. Knockout of Bmal1 gene affects the degree of phosphorylation of Akt and Erk proteins, and the level of ROS production, resulting in a decrease in the protective effects of H2S. Conclusion The expression level of Bmal1 has effects on the function of cardiomyocytes such as ROS production. The potential mechanism by which H2S regulates clock genes may be related to the effect of clock genes on protein phosphorylation levels in ischemic cardiomyocytes.