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Prunella vulgaris has been widely used in the folk medicine of Northeastern Asian countries for the treatment of acute liver injury and infectious hepatitis. In the present study, the protective effect of aqueous extract from P. vulgaris was investigated on carbon tetrachloride-induced hepatic fibrosis in vivo. Our data showed that the administration of aqueous extract from P. vulgaris at doses of 50, 100, and 200 mg/kg significantly reduced the elevated serum levels of alanine aminotransferase, aspartate aminotransferase, type III precollagen, and hyaluronic acid in rats with hepatic fibrosis. In addition, aqueous extract from P. vulgaris also reduced the incidence of liver lesions and the formation of fibrous septa, and remarkably decreased the serum levels of inflammatory cytokines, platelet derived growth factor, interleukin-4, interleukin-8, and tumor necrosis factor alpha. Furthermore, aqueous extract from P. vulgaris significantly inhibited the activation of hepatic stellate cells by regulating the expression of α smooth muscle actin, transforming growth factor β 1, and smad2 and also decreased the deposition of extracellular matrix proteins via regulating the expressions of tissue inhibitor of metalloproteinase-1, matrix metalloproteinase-2,-13. Real-time polymerase chain reaction further revealed that post-treatment with aqueous extract from P. vulgaris decreased the elevated levels of miR-34a and miR-199a-5p in hepatic fibrosis rats. These results demonstrated that aqueous extract from P. vulgaris alleviates carbon tetrachloride-induced hepatic fibrosis by inhibiting the activation of hepatic stellate cells, promoting collagenolysis and regulating fibrosis-related microRNAs.