Prion protein (PrP) was initially described due to its involvement in transmissible spongiform encephalopathies. It was subsequently demonstrated to be a cell surface molecule involved in many physiological processes, such as vesicle trafficking. Here, we investigated the roles of PrP in the response to insulin and obesity development. Two independent PrP knockout (KO) and one PrP overexpressing (TG20) mouse models were fed high-fat diets, and the development of insulin resistance and obesity was monitored. PrP KO mice fed high-fat diets presented all of the symptoms associated with the development of insulin resistance: hyperglycemia, hyperinsulinemia, and obesity. Conversely, TG20 animals fed high-fat diets showed reduced weight and insulin resistance. Accordingly, the expression of peroxisome proliferator-activated receptor gamma (PPARγ) was reduced in PrP KO mice and increased in TG20 animals. PrP KO cells also presented reduced glucose uptake upon insulin stimulation, due to reduced translocation of the glucose transporter Glut4. Thus, our results suggest that PrP reflects susceptibility to the development of insulin resistance and metabolic syndrome.