Abstract The role of serine/threonine protein phosphatase 5 (PP5) in the development of obesity and insulin resistance associated with high-fat diet-feeding (HFD) was examined using PP5-deficient mice (Ppp5c−/−). Despite similar caloric intake, Ppp5c−/− mice on HFD gained markedly less weight and did not accumulate visceral fat compared to wild-type littermates (Ppp5c+/+). On a control diet, Ppp5c−/− mice had markedly improved glucose control compared to Ppp5c+/+ mice, an effect diminished by HFD. However, even after 10weeks of HFD glucose control in Ppp5c−/− mice was similar to that observed in Ppp5c+/+ mice on the control diet. Thus, PP5 deficiency confers protection against HFD-induced weight gain in mice.