Increased endogenous glucose production (EGP) predominantly from the liver is a characteristic feature of type 2 diabetes, which positively correlates with fasting hyperglycemia. Gluconeogenesis is the biochemical pathway shown to significantly contribute to increased EGP in diabetes. Fructose-1,6-bisphosphatase (FBPase) is a regulated enzyme in gluconeogenesis that is increased in animal models of obesity and insulin resistance. However, whether a specific increase in liver FBPase can result in increased EGP has not been shown. The objective of this study was to determine the role of upregulated liver FBPase in glucose homeostasis. To achieve this goal, we generated human liver FBPase transgenic mice under the control of the transthyretin promoter, using insulator sequences to flank the transgene and protect it from site-of-integration effects. This resulted in a liver-specific model, as transgene expression was not detected in other tissues. Mice were studied under the following conditions: 1) at two ages (24 wk and 1 yr old), 2) after a 60% high-fat diet, and 3) when bred to homozygosity. Hemizygous transgenic mice had an approximately threefold increase in total liver FBPase mRNA with concomitant increases in FBPase protein and enzyme activity levels. After high-fat feeding, hemizygous transgenics were glucose intolerant compared with negative littermates (P < 0.02). Furthermore, when bred to homozygosity, chow-fed transgenic mice showed a 5.5-fold increase in liver FBPase levels and were glucose intolerant compared with negative littermates, with a significantly higher rate of EGP (P < 0.006). This is the first study to show that FBPase regulates EGP and whole body glucose homeostasis in a liver-specific transgenic model. Our homozygous transgenic model may be useful for testing human FBPase inhibitor compounds with the potential to treat patients with type 2 diabetes.