It has been reported that exogenous hydrogen sulfide (H2S) protects against high glucose (HG)-induced cardiac injury and has a modulatory effect on heat shock protein (HSP) and Akt, which play a cardioprotective role. In this study, we examined whether the HSP90/Akt pathway contributes to the protective effects of exogenous H2S against HG-induced injury to H9c2 cardiac cells. Our results revealed that the exposure of H9c2 cardiac cells to 35 mM glucose (HG) for 1 to 24 h decreased the expression of HSP90 and markedly reduced the expression level of phosphorylated (p)-Akt in a time-dependent manner. Co-exposure of the cells to HG and geldanamycin (GA; an inhibitor of HSP90) aggravated the inhibition of the p-Akt expression level by HG. Of note, treatment of the cells with 400 µM NaHS (a donor of H2S) for 30 min prior to exposure to HG significantly attenuated the HG-induced decrease in the expression levels of both HSP90 and p-Akt, along with inhibition of HG-induced cell injury, as indicated by the increase in cell viability and superoxide dismutase (SOD) activity, and by a decrease in the number of apoptotic cells, reactive oxygen species (ROS) generation, as well as by the decreased dissipation of mitochondrial membrance potential (MMP). Importantly, treatment of the cells with GA or LY294002 (an inhibitor of Akt) prior to exposure to NaHS and HG considerably blocked the cardioprotective effects of NaHS against the HG-induced injury mentioned above. On the whole, the findings of this study demonstrate that the inhibition of the HSP90/Akt pathway may be an important mechanism responsible for HG-induced cardiomyocyte injury. We also provide novel evidence that exogenous H2S protects H9c2 cells against HG-induced injury by activating the HSP90/Akt pathway.