This study evaluated the contributions of carboxyl ester lipase (CEL) and pancreatic triglyceride lipase (PTL) in lipid nutrient absorption. Results showed PTL deficiency has minimal effect on triacylglycerol (TAG) absorption under low fat dietary conditions. Interestingly, PTL(-)(/)(-) mice displayed significantly reduced TAG absorption compared with wild type mice under high fat/high cholesterol dietary conditions (80.1 +/- 3.7 versus 91.5 +/- 0.7%, p < 0.05). Net TAG absorption was reduced further to 61.1 +/- 3.8% in mice lacking both PTL and CEL. Cholesterol absorption was 41% lower in PTL(-/-) mice compared with control mice (p < 0.05), but this difference was not exaggerated in PTL(-/-), CEL(-/-) mice. Retinyl palmitate absorption was reduced by 45 and 60% in PTL(-/-) mice (p < 0.05) and PTL(-/-), CEL(-/-) mice (p < 0.01), respectively. After 15 weeks of feeding, the high fat/high cholesterol diet, wild type, and CEL(-/-) mice gained approximately 24 g of body weight. However, body weight gain was 6.2 and 8.6 g less (p < 0.01) in PTL(-/-) and PTL(-/-), CEL(-/-) mice, respectively, despite their consumption of comparable amounts of the high fat/high cholesterol diet. The decrease body weight gain in PTL(-/-) and PTL(-/-), CEL(-/-) mice was attributed to their absorption of fewer calories from the high fat/high cholesterol diet, thereby resulting in less fat mass accumulation than that observed in wild type and CEL(-/-) mice. Thus, this study documents that PTL and CEL serve complementary functions, working together to mediate the absorption of a major portion of dietary fat and fat-soluble vitamin esters. The reduced lipid absorption efficiency due to PTL and CEL inactivation also resulted in protection against diet-induced obesity.