In type 2 diabetes, increased endogenous glucose production (EGP) as a result of elevated gluconeogenesis contributes to hyperglycemia. An increase in glycerol gluconeogenesis has led to the suggestion that, in obese human subjects with type 2 diabetes, there may be an increase in the activity of the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase). The aim of this study was to generate transgenic mice that overexpress human liver FBPase in the liver and assess the consequences to whole-body and hepatic glucose metabolism. FBPase transgenic mice had significantly higher levels of transgene expression in the liver and, as a result, had increased FBPase protein and enzyme activity levels in the liver. This resulted in an increase in the rate of glycerol conversion to glucose but not in EGP. The increased expression of FBPase in the liver did not result in any significant differences compared with littermate control mice in insulin or glucose tolerance. Therefore, it appears that, on its own, an increase in FBPase does not lead to impaired regulation of EGP and hence does not affect whole-body glucose metabolism. This suggests that, for EGP to be increased, other factors associated with obesity are also required.