Liver fibrosis is a major cause of morbidity and mortality worldwide, and the outcome of various chronic liver diseases. Recent studies suggest that aberrant expression of miR-34 is involved in the progression of various liver diseases including hepatocellular carcinoma (HCC). However, it is still poorly understood whether miR-34 mediates the pathogenesis of liver fibrosis. Here, we found that the expression of microRNA-34a-5p (miR-34a-5p) was significantly decreased in patients with hepatitis B virus (HBV)-activated liver fibrosis and HCC, as well as in CC14 (Carbon tetrachloride Tetrachloromethane) induced liver fibrosis model mice. The TGF-β1/Smad3 (Transforming growth factor-β1/Smad3) pathway were significantly augmented in CC14 induced mice compared with normal control, whereas inhibitor of TGF-β1 (SB431542) significantly attenuated liver fibrosis and TGF-β1/Smad3 activation. Administration of the miR-34a-5p mimic de-activated TGF-β1/Smad3 pathway in human hepatic stellate cells (HSC), LX-2. Moreover, the target gene for miR-34a-5p, Smad4, was predicted and verified in LX-2 cells. Taken together, these data demonstrated that overexpression of miR-34 in HSCs ameliorated the development and progression of liver fibrosis by targeting Smad4 and regulating TGF-β1/Smad3 pathway. Strategies targeting miR-34a-5p may be of benefit in the treatment of liver fibrosis.