Several studies in animals and humans have reported beneficial effects of diacylglycerol (DAG) on lipid and energy metabolism. We assessed the effect of DAG versus triacylglycerol (TAG) treatment on total energy expenditure (TEE), total fat oxidation (Fox) and respiratory quotient (RQ), and measured the oxidation rate of each oil using a respiratory chamber and the 13C-stable isotope. Eleven healthy subjects participated in a double-blind, randomized crossover study. Subjects consumed an energy maintenance diet consisting of 55% of total calories from carbohydrate, 15% from protein and 30% from fat during both the 3-day pre-chamber and 36-h chamber period. Fifty percent of the fat was test oil, containing either DAG oil or TAG oil. The oxidation rate of ingested test oils was determined by monitoring 13CO2 excretion in the breath from 13C-labeled diolein or 13C-labeled triolein. There were no significant differences in TEE, RQ and total Fox between the DAG and TAG treatment in the overall analysis. In the subgroup analysis, DAG treatment decreased RQ significantly in subjects with a high fat ratio (HFR) compared to TAG treatment. In addition, ingested diolein oxidation in DAG treatment was significantly faster than triolein oxidation in TAG treatment in the HFR group. Enhanced fat utilization with DAG treatment and rapid oxidation of ingested DAG may, at least in part, explain the greater loss of body weight and body fat related to DAG consumption found in the weight-loss studies.