Excessive absorption of products of dietary fat digestion leads to type 2 diabetes and other obesity-related disorders. Mice deficient in the group 1B phospholipase A₂ (Pla2g1b), a gut digestive enzyme, are protected against diet-induced obesity and type 2 diabetes without displaying dietary lipid malabsorption. This study tested the hypothesis that inhibition of Pla2g1b protects against diet-induced hyperlipidemia. Results showed that the Pla2g1b(-/-) mice had decreased plasma triglyceride and cholesterol levels compared with Pla2g1b(+/+) mice subsequent to feeding a high-fat, high-carbohydrate (hypercaloric) diet. These differences were evident before differences in body weight gains were observed. Injection of Poloxamer 407 to inhibit lipolysis revealed decreased VLDL production in Pla2g1b(-/-) mice. Supplementation with lysophosphatidylcholine, the product of Pla2g1b hydrolysis, restored VLDL production rates in Pla2g1b(-/-) mice and further elevated VLDL production in Pla2g1b(+/+) mice. The Pla2g1b(-/-) mice also displayed decreased postprandial lipidemia compared with Pla2g1b(+/+) mice. These results show that, in addition to dietary fatty acids, gut-derived lysophospholipids derived from Pla2g1b hydrolysis of dietary and biliary phospholipids also promote hepatic VLDL production. Thus, the inhibition of lysophospholipid absorption via Pla2g1b inactivation may prove beneficial against diet-induced hyperlipidemia in addition to the protection against obesity and diabetes.