Simultaneous lipolysis and reesterification form the triacylglycerol/fatty acid (TAG/FA) cycle, a substrate cycle commonly used for thermogenesis. Its rate was measured in vivo by indirect calorimetry and continuous infusion of [2-³H]glycerol and [1-¹⁴C]palmitate, after injection of leptin or vehicle saline in rabbits. Leptin stimulated in vivo lipolysis from 9.66 ± 0.62 to 14.78 ± 0.93 μmol · kg⁻¹ · min⁻¹, the rate of appearance of FA from 20.69 ± 2.14 to 29.03 ± 3.03 μmol · kg⁻¹ · min⁻¹, and TAG/FA cycling from 24.82 ± 1.73 to 37.09 ± 2.49 μmol FA · kg⁻¹ · min⁻¹. This large increase in total cycling was caused by an 85% rise in primary cycling (reesterification without transit in the circulation) and accounted for 14% of the difference in metabolic rate between the controls and the leptin-treated animals. This study shows that leptin causes a strong activation of TAG/FA cycling, lipolysis, and FA oxidation, shifting fuel preference from carbohydrates to lipids. Therefore, the acceleration of substrate cycling is a new mechanism triggered by leptin to increase metabolic rate, besides the known induction of uncoupling proteins.