--- - |2+ Background Previous studies have suggested that exogenous hydrogen sulfide can alleviate the development of diabetic cardiomyopathy (DCM) by inhibiting oxidative stress, inflammation, and apoptosis. However, the underlying mechanism is not fully understood. Nuclear expression and function of the transcription factor Forkhead box protein O (FoxO1) have been associated with cardiovascular diseases, and thus, the importance of FoxO1 in DCM has gained increasing attention. This study was designed to investigate the interactions between hydrogen sulfide (H2S) and nuclear FoxO1 in DCM. Methods Diabetes was induced in adult male C57BL/6J mice by intraperitoneal injection of streptozotocin and was treated with H2S donor sodium hydrosulfide for 12 weeks. The H9C2 cardiomyoblast cell line and neonatal rat cardiomyocytes (NRCMs) were treated with the slow‐releasing H2S donor GYY4137 before high‐glucose (HG) exposure with or without pretreatment with the Akt inhibitor MK‐2206 2HCl. Changes in FoxO1 protein phosphorylation and subcellular localization were determined in H9C2 cells, NRCMs, and cardiac tissues from normal and diabetic mice. Cardiac structure and function in the diabetic mice were evaluated by echocardiography and histological analysis and compared with those in control animals. Results The echocardiographic and histopathological data indicated that exogenous H2S improved cardiac function and attenuated cardiac hypertrophy and myocardial fibrosis in diabetic mice. H2S also improved HG‐induced oxidative stress and apoptosis in cardiac tissue and NRCMs. In addition, H2S induced FoxO1 phosphorylation and nuclear exclusion in vitro and in vivo, and this function was not inhibited by MK‐2206 2HCl. Alanine substitution mutation of three sites in FoxO1‐enhanced FoxO1 transcriptional activity, and subsequent treatment with exogenous H2S could not prevent HG‐induced nuclear retention. Conclusions Our data indicate that H2S is a novel regulator of FoxO1 in cardiac cells and provide evidence supporting the potential of H2S in inhibiting the progression of DCM. - Journal of Cellular Physiology, Volume 233, Issue 12, Page 9786-9798, December 2018.