Defective hepatic glucose metabolism contributes to the pathogenesis of impaired glucose tolerance (IGT). However, the precise mechanism(s) responsible have not been fully elucidated. We aimed to combine stable isotope methodology and metabolomic approaches to define the biochemical events underlying aberrant hepatic glucose metabolism under fasting and post-prandial conditions in high-fat diet (HFD) fed mice. Mice fed a HFD fed displayed fasting hyperglycaemia and hyperinsulinaemia, yet endogenous glucose production (EGP) was normal. However, using 2H2O labelling, we detected that the sources of EGP were altered in HFD mice such that rates of gluconeogenesis were reduced while glycogenolysis was increased. To characterise the defects responsible for hyperglycaemia under post-prandial conditions, stable isotope labelled oral glucose tolerance tests (OGTT) were performed. During the OGTT EGP was modestly and transiently suppressed (↓15% at 30 min). Interestingly, the pattern of EGP suppression did not differ between chow and HFD mice. Rather, defects in glucose disposal were responsible for the HFD-induced IGT. As the liver is responsible for ~30% of meal-derived glucose disposal, we examined whether defects in hepatic intermediary metabolism contribute to the impairment in glucose disposal in HFD mice. Therefore, [U-13C]-glucose was orally administered and dynamic metabolomics performed. Livers from HFD mice exhibited an impaired ability to stimulate glycolytic flux, with defects identified at key proximal steps of glycolysis. Consistent with this, we observed reductions (~50%) in absolute metabolite levels in the glycolytic and the pentose pathways in HFD livers. Interestingly, the expression of gluconeogenic and glycolytic genes did not correlate with glucose fluxes, while activation of insulin signalling was unaltered by HFD. Our findings suggest that defects in hepatic glucose uptake and not production are key determinants of IGT in HFD mice and support the concept that metabolic but not signalling or transcriptional changes underlie these defects.