Following liver injuries, hepatic stellate cells (HSCs) express α-SMA. Mitogen activated protein kinase (MAPK) signaling pathways mediate α-SMA expression in distinct cell types. However, the regulation of α-SMA expression by MAPKs in HSCs has been rarely studied. We aimed to study the role of MAPKs in the activation of HSCs during liver fibrosis. Liver fibrosis of rats was induced by carbon tetrachloride. HSC-T6 cells, murine embryonic fibroblasts, JNK1−/− and JNK2−/− cells were used for in vitro studies. Immunohistochemistry and immunoblot analysis were used. We have found that the expression of JNK and α-SMA co-localized in HSCs during liver fibrosis, but ERK and p38 expressed in macrophages. The expression of α-SMA was up-regulated by JNK1 and JNK2 in non-stress condition. Under TGF-β stimulation, however, the level α-SMA expression was increased by only JNK1, but not significantly changed by JNK2. We suggest that JNKs are responsible for α-SMA regulation, and especially JNK1 has a major role in up-regulation of α-SMA expression in HSCs under stress condition induced by TGF-β during liver fibrosis.