OBJECTIVE The purpose of this study was to examine glucose- and metabolically modulation of insulin secretion by rAAV-mediated gene delivery in vitro and in vivo. METHODS A recombinant adeno-associated virus vector (rAAV) containing a furin-mutated human insulin gene, driven by the rat insulin I promoter, was used in this study. Glucose-responsive secretion of human insulin was determined by treating rAAV-transduced Huh7 human hepatoma cells with varying concentrations of glucose, with or without insulin secretagogues. Glucose- and metabolically modulated secretion of human insulin in the streptozotocin (STZ)-induced diabetic mice was assessed by intrahepatic administration of rAAV-polyethylenimine (PEI) complexes, followed by intraperitoneal glucose tolerance test (IPGTT), with or without theophylline. RESULTS Glucose- and metabolically controlled human insulin secretion was obtained in the rAAV-transduced Huh7 cells. Treatment of STZ-induced diabetic animals with rAAV-polyethylenimine (rAAV-PEI) complexes resulted in production of human insulin and amelioration of hyperglycemia. Co-administration of glucose and theophylline in these animals augmented the secretion of human insulin, demonstrating metabolic modulation of insulin secretion in vivo. Immunohistochemical examination of the liver sections of rAAV-treated mice confirmed the production of human insulin. CONCLUSIONS Glucose- and metabolically controlled hepatic insulin gene therapy was obtained both in vitro and in vivo.