A fine control of the blood glucose level is essential to avoid hyper-or hypo-glycemic shocks associated with many metabolic disorders, including diabetes mellitus and type I glycogen storage disease. Between meals, the primary source of blood glucose is gluconeogenesis and glycogenolysis. In the final step of both pathways, glucose-6-phosphate (G6P) is hydrolyzed to glucose by the glucose-6-phosphatase (G6Pase) complex. Because G6Pase (renamed G6Pase-␣) is primarily expressed only in the liver, kidney, and intestine, it has implied that most other tissues cannot contribute to interprandial blood glucose homeostasis. We demonstrate that a novel, widely expressed G6Pase-related protein, PAP2.8/ UGRP, renamed here G6Pase-, is an acid-labile, vanadate-sensitive, endoplasmic reticulum-associated phosphohydrolase, like G6Pase-␣. Both enzymes have the same active site structure, exhibit a similar K m toward G6P, but the V max of G6Pase-␣ is ϳ6-fold greater than that of G6Pase-. Most importantly, G6Pase- couples with the G6P transporter to form an active G6Pase complex that can hydrolyze G6P to glucose. Our findings challenge the current dogma that only liver, kidney, and intestine can contribute to blood glucose homeostasis and explain why type Ia glycogen storage disease patients, lacking a functional liver/kidney/intestine G6Pase complex, are still capable of endogenous glucose production.