Aspalathin (ASP) can protect H9c2 cardiomyocytes against high glucose (HG)-induced shifts in myocardial substrate preference, oxidative stress, and apoptosis. The protective mechanism of ASP remains unknown. However, as one of possible, it is well known that phytochemical flavonoids reduce oxidative stress via nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activation resulting in up-regulation of antioxidant genes and enzymes. Therefore, we hypothesized that ASP protects the myocardium against HG- and hyperglycemia-induced oxidative damage by up-regulating Nrf2 expression in H9c2 cardiomyocytes and diabetic (db/db) mice, respectively. Using an oxidative stress RT2 Profiler PCR array, ASP at a dose of 1 µM was demonstrated to protect H9c2 cardiomyocytes against HG-induced oxidative stress, but silencing of Nrf2 abolished this protective response of ASP and exacerbated cardiomyocyte apoptosis. Db/db mice and their non-diabetic (db/+) littermate controls were subsequently treated daily for six weeks with either a low (13 mg/kg) or high (130 mg/kg) ASP dose. Compared to nondiabetic mice the db/db mice presented increased cardiac remodeling and enlarged left ventricular wall that occurred concomitant to enhanced oxidative stress. Daily treatment of mice with ASP at a dose of 130 mg/kg for six weeks was more effective at reversing complications than both a low dose ASP or metformin, eliciting enhanced expression of Nrf2 and its downstream antioxidant genes. These results indicate that ASP maintains cellular homeostasis and protects the myocardium against hyperglycemia-induced oxidative stress through activation of Nrf2 and its downstream target genes.