Estrogens are known to play a role in modulating metabolic processes within the body. The Aromatase knockout (ArKO) mice have been shown to harbor factors of Metabolic syndrome with central adiposity, hyperinsulinemia and male-specific hepatic steatosis. To determine the effects of estrogen ablation and subsequent replacement in males on whole body glucose metabolism, three- and six-month-old male ArKO mice were subjected to whole body glucose, insulin and pyruvate tolerance tests and analyzed for ensuing metabolic changes in liver, adipose tissue, and skeletal muscle. Estrogen-deficient male ArKO mice showed increased gonadal adiposity which was significantly reduced upon 17beta-estradiol (E2) treatment. Concurrently, elevated ArKO serum leptin levels were significantly reduced upon E2 treatment and lowered serum adiponectin levels were restored to wild type levels. Three-month-old male ArKO mice were hyperglycemic, and both glucose and pyruvate intolerant. These phenotypes continued through to 6 months of age, highlighting a loss of glycemic control. ArKO livers displayed changes in gluconeogenic enzyme expression, and in insulin signaling pathways upon E2 treatment. Liver triglycerides were increased in the ArKO males only after 6 months of age, which could be reversed by E2 treatment. No differences were observed in insulin-stimulated ex vivo muscle glucose uptake nor changes in ArKO adipose tissue and muscle insulin signaling pathways. Therefore, we conclude that male ArKO mice develop hepatic glucose intolerance by the age of 3 months which precedes the sex-specific development of hepatic steatosis. This can be reversed upon the administration of exogenous E2.