In liver, glucose-6-phosphatase catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate, the final step in the gluconeogenic and glycogenolytic pathways. Mutations in the glucose-6-phosphatase catalytic subunit (G6Pase) give rise to glycogen storage disease (GSD) type 1a, which is characterized in part by hypoglycemia, growth retardation, hypertriglyceridemia, hypercholesterolemia, and hepatic glycogen accumulation. Recently, a novel G6Pase isoform was identified, designated UGRP/G6Pase-β. The activity of UGRP relative to G6Pase in vitro is disputed, raising the question as to whether G6P is a physiologically important substrate for this protein. To address this issue we have characterized the phenotype of UGRP knock-out mice. G6P hydrolytic activity was decreased by ∼50% in homogenates of UGRP-/- mouse brain relative to wild type tissue, consistent with the ability of UGRP to hydrolyze G6P. In addition, female, but not male, UGRP-/- mice exhibit growth retardation as do G6Pase-/- mice and patients with GSD type 1a. However, in contrast to G6Pase-/- mice and patients with GSD type 1a, UGRP-/- mice exhibit no change in hepatic glycogen content, blood glucose, or triglyceride levels. Although UGRP-/- mice are not hypoglycemic, female UGRP-/- mice have elevated (∼60%) plasma glucagon and reduced (∼20%) plasma cholesterol. We hypothesize that the hyperglucagonemia prevents hypoglycemia and that the hypocholesterolemia is secondary to the hyperglucagonemia. As such, the phenotype of UGRP-/- mice is mild, indicating that G6Pase is the major glucose-6-phosphatase of physiological importance for glucose homeostasis in vivo.