Providing a conducive microenvironment is critical to increase survival of transplanted stem cells in regenerative therapy. Hyperglycemia promotes stem cell death impairing cardiac regeneration in the diabetic heart. Understanding the molecular mechanisms of high glucose-induced stem cell death is important for improving cardiac regeneration in diabetic patients. Matrix metalloproteinase-9 (MMP9), a collagenase, is upregulated in the diabetic heart, and ablation of MMP9 decreases infarct size in the non-diabetic myocardial infarction heart. In the present study, we aim to investigate whether MMP9 is a mediator of hyperglycemia-induced cell death in human cardiac stem cells (hCSCs) in vitro. We created MMP9 hCSCs to test the hypothesis that MMP9 mediates hyperglycemia-induced oxidative stress and cell death via apoptosis and pyroptosis in hCSCs, which is attenuated by the lack of MMP9. We found that hyperglycemia induced oxidative stress and increased cell death by promoting pyroptosis and apoptosis in hCSCs, which was prevented in MMP9 hCSCs. These findings revealed a novel intracellular role of MMP9 in mediating stem cell death and provide a platform to assess whether MMP9 inhibition could improve hCSCs survival in stem cell therapy at least in acute hyperglycemic microenvironment.