Abstract: Objective To investigated the effects of bisoprolol pretreatment on hypoxia/reoxygenation (H/R)-induced injury in H9c2 cardiomyocytes. Methods Cultured H9c2 cells were exposed to hypoxia for 6 h followed by reoxygenation for 2 h with or without pretreatments with bisoprolol or bisoprolol +LY294002. The cell survival was measured by MTT assay, and the cell apoptosis and levels of radical oxygen species (ROS) were evaluated with flow cytometry. The protein levels of phosphyorylated AKT and phosphorylated GSK3β in the cells were determined by Western blotting. Results Compared with the normal control cells, the cells exposed to H/R injury showed significantly decreased cell survival and increased cell apoptosis and ROS production; pretreatment of the cells with bisoprolol significantly decreased the cell apoptotic rates and ROS production and obviously enhanced the cell survival and protein levels of p-AKT and p-GSK3β in the exposed cells. The protective effect of bsioprolol against H/R-induced cell injury was significantly attenuated by LY294002. Conclusion Bisoprolol can protect H9c2 cells against H/R-induced injury and oxidative stress by activating PI3K/AKT/Gsk-3β pathway to increase the phosphorylation of AKT and GSK3β and reduce ROS production.