This study aimed to investigate the therapeutic potential of isolated mitochondrial transplantation for the restoration of corneal surface injury in mice after corneal chemical burn. Mitochondria were isolated from mesenchymal stem cells via ultracentrifugation, followed by an assessment of their purity and viability. The internalization of mitochondria by human corneal epithelial cells was tracked using a live fluorescence imaging system. Apoptosis-related markers and mitochondrial function were measured by Western blotting and flow cytometry, respectively. Mitochondrial morphology was examined using confocal laser scanning microscopy. The therapeutic effects of subconjunctival administration of isolated mitochondria in vivo were evaluated by fluorescein sodium staining and histopathological examination of the corneas. Our study demonstrates that corneal epithelial cells possess the capacity to internalize isolated exogenous mitochondria in vitro. Under oxidative stress conditions, recipient cells exhibited an enhanced uptake of exogenous mitochondria. We observed a decrease in apoptosis and a reduction in oxidative stress levels within the recipient cells, as well as a partial restoration of mitochondrial function. Notably, after a single subconjunctival injection, corneal epithelial cells were able to use isolated mitochondria to enhance the repair process in a mouse model of corneal acid burn. Subconjunctival injection of isolated mitochondria promoted the repair of acute corneal burns in mice. The results of our investigation using injection of isolated mitochondria as a treatment modality for corneal chemical burn offers a novel approach to the treatment of ocular disorders associated with mitochondrial dysfunction.