Previous studies have demonstrated dysregulated mitochondrial dynamics in fibrotic livers and hepatocytes. Little is currently known about how mitochondrial dynamics are involved, nor is it clear how mitochondrial dynamics participate in hepatic stellate cell (HSC) activation. In the present study, we investigated the role of mitochondrial dynamics in HSC activation and the underlying mechanisms. We verified that mitochondrial fission was enhanced in human and mouse fibrotic livers and active HSCs. Moreover, increased mitochondrial fission driven by fis1 overexpression could promote HSC activation. Inhibiting mitochondrial fission using mitochondrial fission inhibitor-1 (Mdivi-1) could inhibit activation and induce apoptosis of active HSCs, indicating that increased mitochondrial fission is essential for HSC activation. Mdivi-1 treatment also induced apoptosis in active HSCs in vivo and thus ameliorated CCl4-induced liver fibrosis. We also found that oxidative phosphorylation (OxPhos) was increased in active HSCs, and OxPhos inhibitors inhibited activation and induced apoptosis in active HSCs. Moreover, increasing mitochondrial fission upregulated OxPhos, while inhibiting mitochondrial fission downregulated OxPhos, suggesting that mitochondrial fission stimulates OxPhos during HSC activation. Next, we found that inhibition of oxidative stress using mitoquinone mesylate (mitoQ) and Tempol inhibited mitochondrial fission and OxPhos and induced apoptosis in active HSCs, suggesting that oxidative stress contributes to excessive mitochondrial fission during HSC activation. In conclusion, our study revealed that oxidative stress contributes to enhanced mitochondrial fission, which triggers OxPhos during HSC activation. Importantly, inhibiting mitochondrial fission has huge prospects for alleviating liver fibrosis by eliminating active HSCs.