Liver fibrosis is a pathological wound-healing response caused by chronic liver damage. Mitochondria regulate hepatic energy metabolism and oxidative stress. Accumulating evidence has revealed that increased mitochondrial oxidative stress contributes to the activation of fibrogenesis. However, the roles and underlying mechanisms of mitochondrial oxidative stress in liver fibrosis remain unknown. In this study, C57BL/6 mice were used to establish a model of liver fibrosis via oral gavage with CCl4 treatment for 8 weeks. Furthermore, intervention experiments were achieved by CCl4 combined with the intraperitoneal injection of mitoquinone mesylate (mitoQ). We demonstrated that the chronic CCl4 exposure resulted in severe hepatic fibrogenesis and significantly promoted the production of reactive oxygen species (ROS) and mitochondrial abnormalities. Besides, JNK/YAP pathway was also activated. By contrast, the administration of mitoQ markedly inhibited the expression of pro-fibrogenic transforming growth factor-β as well as type I collagen. The antifibrotic effects of mitoQ were also confirmed by hematoxylin and eosin staining and Sirius red staining. Moreover, mitoQ substantially reduced CCl4-induced mitochondrial damage and the release of ROS. Further studies suggested that this protection against liver fibrosis was mechanistically related to the inhibition of phosphorylation of JNK and the nuclear translocation of YAP. In conclusion, these findings revealed that mitoQ attenuated liver fibrosis by inhibiting ROS production and the JNK/YAP signaling pathway. Selective targeting JNK/YAP may serve as a therapeutic strategy for retarding progression of chronic liver disease.