Diacylglycerol (DAG) production by glycerolysis of glycerol with tuna oil was performed using lipases from Rhizomucor miehei (Lipozyme RMIM) and Candida antarctica (Novozyme 435). Lipozyme RMIM caused a clear estrific positional specificity in HPLC analysis and then the Lipozyme RMIM was chosen for the production of DAG. Moreover, the reaction parameter for DAG synthesis was determined by measuring mole ratio of glycerol/tuna oil, amount of enzyme, temperature and water contents. The optimal mole ratio for glycerol/tuna oil was established to be 3:1. Optimal conditions of lipase, water and temperature were 10%, 10% and 35 C, respectively. Therefore, we have synthesized DAG from tuna oil under these optimal conditions and investigated the effect of the synthesized DAG on body weight and plasma biochemical markers of obesity in C57BL/6J mice. The consumption of DAG diet has effectively lessened body weight gain and final plasma total cholesterol, triacylglycerol and glucose levels compared to the high triacylglycerol (TAG) group. (C) 2010 Elsevier Ltd. All rights reserved.