Myofibroblasts are important mediators of fibrogenesis, thus blocking fibroblast to myofibroblast differentiation (FMD) may be an effective strategy to treat pulmonary fibrosis (PF). Previously we reported that HDAC4 activity is necessary for TGF-β1-induced human lung FMD. Here we show that TGF-β1 increases NOX4 mRNA and protein expression in normal human lung fibroblasts (NHLFs) and causes nuclear export of HDAC4. Application of the NOX family inhibitor, diphenyleneiodonium chloride (DPI), reduces TGF-β1-induced HDAC4 nuclear export, expression of the myofibroblast marker α-smooth muscle actin (α-SMA), and α-SMA fiber formation. Inhibition of HDAC4 nucleus to cytoplasm translocation using leptomycin B (LMB) had little effect on α-SMA expression, but blocked α-SMA fiber formation. A co-immunoprecipitation assay showed that HDAC4 associates with α-SMA. Moreover LMB abolishes TGF-β1-induced α-SMA fiber formation and cell contraction. Relevant to human pulmonary fibrosis, IPF specimens showed significantly higher NOX4 RNA expression and scant HDAC4 staining within nuclei of fibroblast foci myofibroblasts. Taken together, these results indicate that ROS promote TGF-β1-mediated myofibroblast differentiation and HDAC4 nuclear export. The physical association of HDAC4 with α-SMA suggests that HDAC4 has a role in regulating the α-SMA cytoskeleton arrangement.