Berberine is an isoquinoline alkaloid with anti-diabetic properties. Despite the central role of liver and thus hepaticmitochondria inwhole-bodymetabolism, berberine effects on hepaticmitochondrial function in an obesity model are still unknown. Here, we demonstrate that berberine treatment recovers mitochondrial efficiency when altered by a high-fat feeding.Mitochondria isolated from the liver of high-fat fed rats exhibited decreased capacity to accumulate calcium and impaired oxidative phosphorylation (OXPHOS) capacity, as shown by impaired mitochondrialmembrane potential, oxygen consumption and cellular ATP levels. Interestingly, the recovery of mitochondrial function by berberine was associated with an increased activity of the mitochondrial sirtuin 3 (SirT3). In conclusion, berberine potent protective effects against metabolic syndrome may rely on increasing mitochondrial SirT3 activity, normalizing mitochondrial function and preventing a state of energetic deficit caused by impaired OXPHOS. ; JST, FVD, APG and ATV were recipients of a Fundação para a Ciência e a Tecnologia PhD scholarship (SFRH/BD/38467/2007, SFRH/BD/38372/ 2007, SFRH/BD/44674/2008 and SFRH/BD/44796/2008, respectively). This project was supported by a FCT grant PTCD/SAU-OSM/72443/ 2006.