Objective：The aim of this study was to investigate the mechanism underlying transforming growth factor-β1 (TGF-β1) induced differentiation of bone marrowderived mesenchymal stem cells (BMSCs)into myofibroblasts.Methods:Primary mouse BMSCs were isolated from bone marrow by flushing the tibias and femurs of mice, and passage 3 to passage 5 of BMSCs were used in the experiments. BMSCs differentiation into myofibroblast was induced by different doses of TGF-β1. In addition, reactive oxygen species (ROS) inhibitor (N-acetylcysteine, NAC) was added to test its effect on the action of TGF-β1. Expressions of BMSCs differentiation parameters, α-smooth muscle actin (α-SMA), collagen α1(Ⅰ) ［Col α1(Ⅰ)］ and collagen α1(Ⅲ) ［Col α1(Ⅲ)］ were measured by real-time quantitative PCR (RT-qPCR) and Western blot analysis. BMSCs were preloaded for 15 min with 2’, 7’-dichlorohydro-fluorescein diacetate (DCFH-DA), then stimulated with TGF-β1 for different times, and fluorescence of ROS was measured using high content analysis. Results:TGF-β1 stimulated differentiation of BMSCs into myofibroblasts and up-regulated expression of α-SMA, Col α1(Ⅰ) and Col α1(Ⅲ) in a dose-dependent manner, which blocked by ROS inhibitor NAC. In addition, TGF-β1 could induce a significant rapid and transient increase in ROS production in BMSCs, and the effect of TGF-β1 on ROS production was peaked at 30 min.Conclusion:TGF-β1 induced differentiation of BMSCs into myofibroblasts via production of ROS.