Aquaporin-9 (AQP9) is an aquaglyceroporin membrane channel shown biophysically to conduct water, glycerol, and other small solutes. Because the physiological role/s of AQP9 remain undefined and the expression sites of AQP9 remain incomplete and conflicting, we generated AQP9 knockout mice. In the absence of physiological stress, knockout mice did not display any visible behavioral or severe physical abnormalities. Immunohistochemical analyses using multiple antibodies revealed AQP9 specific labeling in hepatocytes, epididymis, vas deferens, and in epidermis of wild type mice, but a complete absence of labeling in AQP9^−/− mice. In brain, no detectable labeling was observed. Compared with control mice, plasma levels of glycerol and triglycerides were markedly increased in AQP9^−/− mice, whereas glucose, urea, free fatty acids, alkaline phosphatase, and cholesterol were not significantly different. Oral administration of glycerol to fasted mice resulted in an acute rise in blood glucose levels in both AQP9^−/− and AQP9^+/− mice, revealing no defect in utilization of exogenous glycerol as a gluconeogenic substrate and indicating a high gluconeogenic capacity in nonhepatic organs. Obese Lepr^db/Lepr^db AQP9^−/− and obese Lepr^db/Lepr^db AQP9^+/− mice showed similar body weight, whereas the glycerol levels in obese Lepr^db/Lepr^db AQP9^−/− mice were dramatically increased. Consistent with a role of AQP9 in hepatic uptake of glycerol, blood glucose levels were significantly reduced in Lepr^db/Lepr^db AQP9^−/− mice compared with Lepr^db/Lepr^db AQP9^+/− in response to 3 h of fasting. Thus, AQP9 is important for hepatic glycerol metabolism and may play a role in glycerol and glucose metabolism in diabetes mellitus.