Kruppel-like Factor 4 (KLF4), a target gene of miR-145, can negatively regulate lung fibrosis. However, the potential role of KLF4 and miR-145 in hepatic stellate cells (HSCs) activation or in hepatic fibrosis keeps unclear. This study aims to characterize miR-145 and KLF4 in activated HSCs and liver cirrhotic, and the underlying molecular basis. miR-145 was significantly up-regulated, while KLF4 was dramatically down-regulated during the activation of rat primary HSCs and TGF-beta treated HSCs. Furthermore, miR-145 mimics induced and inhibition of miR-145 reduced a-SMA and COL-I expression in primary HSCs. Additionally, the mRNA and protein levels of KLF4 in the liver of cirrhotic patients and rats were significantly down-regulated. alpha-SMA and COL-I were increased after inhibition of KLF4 by specific shRNA in primary HSCs. Forced KLF4 expression led to a reduction of a-SMA and COL-I expression in HSCs. miR-145 promotes HSC activation and liver fibrosis by targeting KLF4.