(-)-Hydroxycitrate (HCA) might promote weight maintenance by limiting the capacity for de novo lipogenesis (DNL). It was investigated whether HCA may reduce DNL in humans during a persistent excess of energy intake as carbohydrate. In a double-blind, placebo-controlled, randomized and cross-over design, 10 sedentary lean male subjects (mean+/-S.D., age: 24+/-5 years, BMI: 21.8+/-2.1 kg/m2) performed a glycogen depletion exercise test followed by a 3-day high-fat diet (F/CHO/P, 60/25/15% energy; 100% of energy expenditure (EE)) and a 7-day high-CHO diet (F/CHO/P, <5/>85/10% energy; 130-175% of EE; overfeeding). During overfeeding, they ingested 3 x 500 mg/day HCA or placebo (PLA). Each intervention ended with a 60-h stay in the respiration chamber (days 9 and 10). Body weight increased during overfeeding (mean+/-S.E., HCA: 2.9+/-0.2 kg, PLA: 2.8+/-0.2 kg). Respiratory quotient (RQ) was >1.00 in all subjects indicating that DNL was present. On day 9, 24-h EE was lower with HCA compared to PLA (P < 0.05). On day 10, resting metabolic rate and RQ during night were lower (P < 0.01 and P < 0.05, respectively). Non-protein RQ, fat balance and net fat synthesis as DNL tended to be lower (P < 0.1) with HCA compared to PLA indicating lower DNL; activity-induced EE was higher with HCA (P < 0.05) indicating the urge to eliminate the excess of energy ingested. We conclude that an experimental condition resulting in DNL in humans was created and that treatment with HCA during overfeeding with carbohydrates may reduce DNL.