Mice lacking acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), a key enzyme in triglyceride synthesis, have increased energy expenditure and therefore are resistant to obesity. Because ambient temperature can significantly affect energy expenditure in mice, we undertook these studies to determine the effects of different ambient temperatures on energy expenditure, food intake, and thermoregulation in DGAT1-deficient [Dgat1(−/−)] mice.Dgat1(−/−) mice had increased energy expenditure irrespective of changes in the ambient temperature. Although core temperature was normal, surface temperature was increased inDgat1(−/−) mice, most likely reflecting an active mechanism to dissipate heat from increased thermogenesis.Dgat1(−/−) mice had increased food intake at baseline, and this hyperphagia became more pronounced upon exposure to cold. When fasted in a cold environment, Dgat1(−/−) mice developed hypothermia, which was associated with hypoglycemia. These results suggest that the hyperphagia in Dgat1(−/−) mice is a secondary mechanism that compensates for the increased utilization of fuel substrates. Our findings offer insights into the mechanisms of hyperphagia and increased energy expenditure in a murine model of obesity resistance.