The G6PC1, G6PC2 and G6PC3 genes encode distinct glucose-6-phosphatase catalytic subunit (G6PC) isoforms. In mice, germline deletion of G6pc2 lowers fasting blood glucose (FBG) without affecting fasting plasma insulin (FPI) while, in isolated islets, glucose-6-phosphatase activity and glucose cycling are abolished and glucose-stimulated insulin secretion (GSIS) is enhanced at submaximal but not high glucose. These observations are all consistent with a model in which G6PC2 regulates the sensitivity of GSIS to glucose by opposing the action of glucokinase. G6PC2 is highly expressed in human and mouse islet beta cells however, various studies have shown trace G6PC2 expression in multiple tissues raising the possibility that G6PC2 also affects FBG through non-islet cell actions. Using real-time PCR we show here that expression of G6pc1 and/or G6pc3 are much greater than G6pc2 in peripheral tissues, whereas G6pc2 expression is much higher than G6pc3 in both pancreas and islets with G6pc1 expression not detected. In adult mice, beta cell-specific deletion of G6pc2 was sufficient to reduce FBG without changing FPI. In addition, electronic health record-derived phenotype analyses showed no association between G6PC2 expression and phenotypes clearly unrelated to islet function in humans. Finally, we show that germline G6pc2 deletion enhances glycolysis in mouse islets and that glucose cycling can also be detected in human islets. These observations are all consistent with a mechanism by which G6PC2 action in islets is sufficient to regulate the sensitivity of GSIS to glucose and hence influence FBG without affecting FPI.