Diabetic cardiomyopathy (DCM) has become a major cause of diabetes-related morbidity and mortality. Increasing evidences have proved that hydrogen sulfide (H2S) fulfills a positive role in regulating diabetic myocardial injury. The present study was designed to determine whether GYY4137, a novel H2S-releasing molecule, protected H9c2 cells against high glucose (HG)-induced cytotoxicity by activation of the AMPK/mTOR signal pathway. H9c2 cells were incubated in normal glucose (5.5 mM), 22, 33, and 44 mM glucose for 24 h to mimic the hyperglycemia in DCM in vitro. Then we added 50, 100, and 200 μM GYY4137, and measured the cell viability, lactate dehydrogenase (LDH) enzyme activity, and mitochondrial membrane potential (MMP). 0.5 mM 5-amino-4-imidazole-carboxamide riboside (AICAR, an AMPK activator) and 1 mM adenine 9-β-D-arabinofuranoside (Ara-A, an AMPK inhibitor) were used to identity whether the AMPK/mTOR signal pathway was involved in GYY4137-mediated cardioprotection. We demonstrated that HG decreased cell viability and increased LDH enzyme activity in a concentration-dependent manner. 33 mM HG treatment for 24 h was chosen as our model group for further study. Both 100 and 200 μM GYY4137 treatments significantly attenuated HG-induced cell viability decrement, LDH enzyme activity increase, and MMP collapse. AICAR had similar effects to GYY4137 treatment while Ara-A attenuated GYY4137-mediated cardioprotection. Importantly, both GYY4137 and AICAR increased AMPK phosphorylation and decreased mTOR phosphorylation compared with the HG model group while Ara-A attenuated GYY4137-mediated AMPK phosphorylation increase and mTOR phosphorylation decrement. In conclusion, we propose that GYY4137 likely protects against HG-induced cytotoxicity by activation of the AMPK/mTOR signal pathway in H9c2 cells.