Aim To study the effect of levosimendan on apoptosis of H9c2 cells under hypoxia/reoxygenation (H/R) condition and related mechanisms. Methods H9c2 cells were cultured in vitro, and the cells were divided into blank control group, H/R group, low-dose levosimendan group, medium-dose levosimendan group, and high-dose levosimendan group. Proliferation of H9c2 cells was detected by methyl thiazolyl tetrazolium assay; Apoptosis of H9c2 cells was detected by flow cytometry; Superoxide dismutase (SOD) kit and malondialdehyde (MDA) kit were used to detect SOD activity and MDA content respectively; Reactive oxygen species (ROS) level was detected by fluorescent probe method; Proliferating cell nuclear antigen (PCNA), B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), phosphate and tension homology deleted on chromsome ten (PTEN) and phosphoinositide 3 kinase (PI3K)/serine threonine kinase (Akt) signaling pathway protein expression were detected by Western blot. Results Compared with the blank control group, the proliferation inhibition rate, apoptosis rate, MDA content, ROS level and PTEN protein expression of H9c2 cells in H/R group were significantly increased (P＜0.05), PCNA, Bcl-2, p-PI3K/PI3K, p-Akt/Akt were significantly decreased, and Bax was significantly increased (P＜0.05). Compared with the H/R group, the proliferation inhibition rate, apoptosis rate, MDA content, ROS level and PTEN protein expression of H9c2 cells in the low-dose levosimendan group, the middle-dose group and the high-dose levosimendan group were significantly decreased (P＜0.05), PCNA, Bcl-2, p-PI3K/PI3K, p-Akt/Akt were significantly increased, and Bax was significantly decreased (P＜0.05); And the higher the dose of levosimendan, the more obvious the corresponding changes of the above indicators. Conclusion Levosimendan can promote cell proliferation, and inhibit cell oxidative stress and apoptosis in H9c2 cells under H/R conditions possibly by inhibiting PTEN and activating the PI3K/Akt signaling pathway.