Impaired hepatic glucose metabolism is characterised by reduced suppression of endogenous glucose production (EGP) as well as reduced glucose uptake and storage. It is a primary defect in the development of insulin resistance and glucose intolerance, however, the precise mechanism(s) are not fully understood. We aimed to combine stable isotope methodology and metabolomic approaches to obtain a detailed understanding of the biochemical events underlying aberrant hepatic glucose metabolism under dynamic conditions in vivo. To examine EGP and the contribution of gluconeogenesis (GNG) and glycogenolysis (GLY), mice fed a chow or high fat diet (HFD) were administered 2H2O and 2H positional enrichment in glucose was studied during an oral glucose tolerance test (OGTT). The HFD impaired glucose tolerance and resulted in hyperinsulinemia during the OGTT. Basal EGP was elevated in HFD mice and remained higher throughout the OGTT. Basally, the fractional contribution of GNG to EGP was lower in HFD mice (92.5% vs 89% for chow and HFD, P<0.05) while the contribution from GLY was increased (7% vs. 11% for chow and HFD, P<0.05). The OGTT suppressed EGP by ~40% in both diets, with the fractional contribution from GNG only being modestly reduced in chow mice. Despite this, GNG remained active during the OGTT (~90% of EGP). GNG gene expression and activation of insulin signalling was unaltered by HFD or OGTT. To define the defects in intracellular hepatic glucose metabolism, [U-13C]-glucose was orally administered to chow and HFD mice and metabolomic flux profiling performed. Livers from HFD mice exhibited an inability to stimulate glycolytic flux, with defects identified at key proximal steps of glycolysis and the pentose phosphate cycle. Our findings support the concept that metabolic but not signalling or transcriptional defects underlie HFD-induced glucose intolerance. These data challenge the view that defective insulin signalling impairs glucose metabolism and highlights the need to perform experiments under dynamic physiologically relevant conditions in vivo.