Increasing evidence suggests that brown adipose tissue (BAT) plays an important role in obesity and related diseases. Great progress has been made in identifying positive regulators that activate adipocyte thermogenesis, but negative regulatory signaling of thermogenesis remains poorly understood. Here, we evaluated the potential effects of aspartate on the BAT function. We found that the circulating aspartate level is positively associated with metabolic syndrome and obesity in adults. Acute cold exposure significantly increases BAT aspartate as well as other amino acid levels in mice. In this regard, we speculate that aspartate may play a role in regulating the BAT function and systemic energy homeostasis. To verify the hypothesis, we altered aspartate availability to explore the effects on adipose tissue metabolism. Supplementation of aspartate exogenously inhibits the thermogenic gene expression and cold tolerance in mice. Intriguingly, aspartate bioavailability inhibits mitochondrial biosynthesis essentially through the suppression of mechanistic targeting of the AMPK cascade. Therefore, an evaluation of whether a diet deficient in aspartate will increase oxidative phosphorylation in the mitochondria to reestablish aspartate levels and therefore increase the energy expenditure will be interesting because these effects can prevent or ameliorate the development of obesity.