Objective: To investigate the differential expression and the effect of apolipoprotein M (ApoM) on H9C2 rat cardiomyocyte injury induced by hypoxia/reoxygenation (H/R). Methods: Using the AnaeroPack System, we established the H/R injury model of H9C2 rat cardiomyocytes. CCK-8 cell viability assay, cell apoptosis and caspase-3 activity assay were used to detect the difference of cell proliferation and apoptosis after H/R injury. Furthermore, the expression levels of ApoM and sphingosine-1-phosphate receptor 1 (S1PR1) were detected. H9C2 cardiomyocytes overexpressing ApoM (ApoM-OE) were established. CCK-8 assay was used to detect the survival rate of cardiomyocytes, the colorimetric method was applied to detect the activities of lactate dehydrogenase (LDH) and superoxide dismutase (SOD) in the cell culture supernatant, immunoblotting was used to determine the expression levels of cleaved caspase-3 and caspase-3, and the role of ApoM in H/R-induced cardiomyocyte injury was analyzed. Results: The H9C2 cardiomyocyte viability decreased gradually after H/R, while apoptosis rate and caspase-3 activity increased after H/R. It confirmed that the H/R injury model of H9C2 rat cardiomyocytes was successfully established through AnaeroPack System. The mRNA expression levels of ApoM and S1PR1 were upregulated in cardiomyocytes after H/R (both P<0.01). Compared with the NC group, the cell survival rate, HDL activity, SOD activity and relative expression level of cleaved caspase-3 in the ApoM-OE group were no significant difference (P>0.05). Conclusion: The expression levels of ApoM and S1PR1 were upregulated in H9C2 after H/R, but ApoM had no significant protective effect on H9C2 rat cardiomyocytes injured by H/R.