Group 1B phospholipase A₂ (PLA₂) is an abundant lipolytic enzyme that is well characterized biochemically and structurally. Because of its high level of expression in the pancreas, it has been presumed that PLA₂ plays a role in the digestion of dietary lipids, but in vivo data have been lacking to support this theory. Our initial study on mice lacking PLA₂ demonstrated no abnormalities in dietary lipid absorption in mice consuming a chow diet. However, the effects of PLA₂ deficiency on animals consuming a high-fat diet have not been studied. To investigate this, PLA₂^+/+ and PLA₂^/ mice were fed a western diet for 16 wk. The results showed that PLA₂^/ mice were resistant to high-fat diet-induced obesity. This observed weight difference was due to decreased adiposity present in the PLA₂^/ mice. Compared with PLA₂^+/+ mice, the PLA₂^/ mice had 60% lower plasma insulin and 72% lower plasma leptin levels after high-fat diet feeding. The PLA₂^/ mice also did not exhibit impaired glucose tolerance associated with the development of obesity-related insulin resistance as observed in the PLA₂^+/+ mice. To investigate the mechanism by which PLA₂^/ mice exhibit decreased weight gain while on a high-fat diet, fat absorption studies were performed. The PLA₂^/ mice displayed 50 and 35% decreased plasma [³H]triglyceride concentrations 4 and 6 h, respectively, after feeding on a lipid-rich meal containing [³H]triolein. The PLA₂^/ mice also displayed increased lipid content in the stool, thus indicating decreased fat absorption in these animals. These results suggest a novel role for PLA₂ in the protection against diet-induced obesity and obesity-related insulin resistance, thereby offering a new target for treatment of obesity and diabetes.