The present study was conducted to evaluate the therapeutic efficacy of Rhinacanthus nasutus (R. nasutus) on mitochondrial and cytosolic enzymes in streptozotocin-induced diabetic rats. The rats were divided into five groups with 6 rats in each group. The methanolic extract of R. nasutus was orally administered at a dose of 200 mg/kg/day, and glibenclamide was administered at a dose of 50 mg/kg/day. All animals were treated for 30 days and were sacrificed. The activities of both intra- and extramitochondrial enzymes including glucose-6-phosphate dehydrogenase (G6PDH), succinate dehydrogenase (SDH), glutamate dehydrogenase (GDH), and lactate dehydrogenase (LDH) were measured in the livers of the animals. The levels of G6PDH, SDH, and GDH were significantly reduced in the diabetic rats but were significantly increased after 30 days of R. nasutus treatment. The increased LDH level in diabetic rats exhibited a significant reduction after treatment with R. nasutus. These results indicate that the administration of R. nasutus altered the activities of oxidative enzymes in a positive manner, indicating that R. nasutus improves mitochondrial energy production. Our data suggest that R. nasutus should be further explored for its role in the treatment of diabetes mellitus.