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 PLA(2)(-/-) mice exhibit decreased weight gain while on a high-fat diet, fat absorption studies were performed. The PLA(2)(-/-) mice displayed 50 and 35% decreased plasma [(3)H]triglyceride concentrations 4 and 6 h, respectively, after feeding on a lipid-rich meal containing [(3)H]triolein. The PLA(2)(-/-) mice also displayed increased lipid content in the stool, thus indicating decreased fat absorption in these animals. These results suggest a novel role for PLA(2) in the protection against diet-induced obesity and obesity-related insulin resistance, thereby offering a new target for treatment of obesity and diabetes.