Myofibroblast differentiation is a key process in the pathogenesis of fibrotic diseases. Transforming growth factor-β1 (TGFβ1) is a powerful inducer of myofibroblast differentiation and is implicated in pathogenensis of tissue fibrosis. This study was undertaken to determine the role of mitochondrial deacetylase, SIRT3 in TGF-β1-induced myofibroblast differentiqtion in vitro and lung fibrosis in vivo. Treatment of human lung fibroblasts with TGFβ1 resulted in increased expression of fibrosis markers, smooth muscle alpha-actin (α-SMA), collagen-1 and fibronectin. TGF-β1 treatment also caused depletion of endogenous SIRT3, which paralleled with increased production of reactive oxygen species (ROS), DNA damage and subsequent reduction in levels of 8-oxoguanine DNA glycosylase (OGG1), an enzyme that hydrolyzes oxidized-guanine (8-Oxo-dG), and thus protects DNA from oxidative damage. Overexpression of SIRT3 by adenovirus-mediated transduction reversed the effects of TGF-β1 on ROS production and mitochondrial DNA damage, and inhibited TGF-β1-induced myofibroblast differentiation. To determine the anti-fibrotic role of SIRT3 in vivo, we used the bleomycin-induced mouse model of pulmonary fibrosis. Compared to wild-type controls, SIRT3-KO mice showed exacerbated fibrosis after intratracheal instillation of bleomycin. Increased lung fibrosis was associated with decreased levels of OGG1 and concomitant accumulation of 8-Oxo-dG and increased mitochondrial DNA damage. In contrast, the transgenic mice with whole body SIRT3 overexpression were protected from bleomycin-induced mtDNA damage and development of lung fibrosis. These data demonstrate a critical role of SIRT3 in the control of myofibroblast differentiation and lung fibrosis.