The activation of nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated signaling pathway has been involved in the mechanisms of a variety of protective agents against cellular oxidative stress. We recently demonstrated that Dendrobium nobile Lindl. alkaloids (DNLA), the active ingredients of Dendrobium, protects mice from CCl₄-induced liver injury, dependent on the Nrf2 signaling pathway. The present study was aimed to determine whether the protection against mitochondrial oxidative damage plays a role in the mode of action of DNLA on CCl₄-induced liver injury, and to further investigate whether the DNLA-conferred mitochondrial beneficial effects is dependent on the activation of Nrf2 signaling. The CCl₄-induced acute liver injury model was employed in both wild-type (WT) and Nrf2-knockout (Nrf2^−/−) mice. The results showed that in WT mice DNLA reduced CCl₄-induced liver injury, accompanied by a significant reduction in CCl₄-induced mitochondrial oxidative stress as evidenced by a decrease in mitochondrial H₂O₂ content and MDA production, and a marked increase in GSH level and Mn-SOD activity. However, these protective effects were significantly attenuated in Nrf2^−/− mice. Furthermore, the administration of DNLA improved mitochondrial oxygen consumption, elevated ATP production, and decreased CCl₄-induced apoptosis in the WT mice, whereas the DNLA-mediated protections on mitochondrial function were diminished in the Nrf2 null mice. These results demonstrate that the improvement of mitochondrial oxidative stress and mitochondrial dysfunction is implicated in the mechanism of DNLA-mediated protection on CCl₄-induced liver injury, and this DNLA-modulated mode of action is dependent on the activation of Nrf2 signaling pathway.