Background Insulin self-administration is burdensome and can produce dangerous hypoglycemia. Insulin gene therapy may improve and simplify the treatment of diabetes mellitus. In rats, metabolically responsive hepatic insulin gene therapy (HIGT) delivered by adenovirus normalizes random blood sugars but with a limited duration. To prolong glycemic control, we delivered a metabolically regulated insulin transgene by adeno-associated virus (AAV). Methods We administered increasing doses of self-complementary (SC), pseudotyped AAV8 expressing the (GlRE)(3)BP1-2xfur insulin transgene to streptozotocin-diabetic CD-1 mice, and monitored blood sugar and body weight. We also compared responses to intraperitoneal glucose and chow withdrawal, assessed for viral genomes in liver by Southern blotting, and measured hepatic glycogen. Results Glucose lowering required the combination of SC genomes and AAV capsid pseudotyping. HIGT controlled glycemia in diabetic mice (DM) for >1 year. However, glycemic responses were variable. Approximately 30% of mice succumbed to hypoglycemia, and approximately 30% of mice again became hyperglycemic. During an intraperitoneal glucose tolerance test, blood sugars declined to normal within 180 min in HIGT-treated DM compared to 90 min in control mice. Hypoglycemia was common among HIGT-treated mice during a 24-h fast. However, HIGT mice lost less weight than either diabetic or nondiabetic controls as a result of increased water intake. HIGT treatment reduced the hepatic glycogen content of fed mice. Conclusions Our studies demonstrate the possibility for long-term glycemic correction following AAV-mediated HIGT in mice. However, the dose-response relationship is irregular, and metabolic responsiveness may be less than that observed in rats. Copyright (C) 2015 John Wiley & Sons, Ltd.