The purpose of the present study was to document the metabolism of glucose and its responsiveness to insulin in isolated pancreatic acini from rats fed either a low- or high-fat diet. The different steps investigated were labeled glucose oxidation, lactate production, hexokinase activity, and glucose transport, which was assessed by using both 3-O-methylglucose and 2-deoxyglucose. The acinar capacity to metabolize glucose (sum of CO2 plus lactate) was decreased by 50% by feeding the rats a high-fat diet. The impairment of glucose metabolism could not be explained by a defect in the glucose phosphorylation step because hexokinase activity was not changed in isolated acini from rats fed a high-fat diet. The effect of a high-fat diet was entirely accounted for by a reduction in the glucose transport rate that was achieved through a decrease in glucose transport Vmax with no change in Km. We could not detect any effect of insulin on glucose metabolism or 2-deoxyglucose uptake, whatever the diet composition. This work establishes that a high-fat diet, known to markedly alter pancreatic exocrine enzymes, also induces a large decrease in acinar glycolytic flux, raising the question of the relation between these two sets of adaptive changes.