Background & aims: Glucose-6 phosphatase (Glc6Pase) is the last enzyme of gluconeogenesis and glycogenolysis, previously assumed to be expressed in the liver and kidney only, conferring on both tissues the capacity to produce endogenous glucose in blood. Methods: Using Northern blotting and reverse-transcription polymerase chain reaction and a highly specific Glc6Pase assay, we studied expression of the Glc6Pase gene in human and in rat tissues (fasted and diabetic). Results: The Glc6Pase gene is expressed in the duodenum and jejunum in normal fed rats and in the duodenum, jejunum, and ileum in humans. The Glc6Pase messenger RNA (mRNA) abundance was increased eightfold and sixfold in the duodenum and jejunum of streptozotocin diabetic rats. It was normalized in both tissues after 10 hours of insulin treatment. Glc6Pase activity was increased by 300% in the duodenum and jejunum in diabetic rats compared with normal rats. The Glc6Pase mRNA abundances and enzymatic activities were increased in a similar manner in both tissues in 48-hour-fasted rats. Normalization of mRNA abundance was achieved after refeeding for 7 hours. In addition, Glc6Pase mRNA and activity were also expressed in the ileum during fasting in rats. Conclusions: These data show that the small intestine has the ability to release endogenous glucose and strongly suggest that its contribution to systemic glucose production might be increased in situations of insulinopenia (type 1 diabetes) and insulin resistance (type 2 diabetes and others).