Background and Aims: Acute kidney injury (AKI) is independently associated with high mortality after major trauma. Because mitochondrial dysfunction in renal tubular cells is a critical early subcellular change in AKI, this study proposed that administration of freshly harvested viable mitochondria could ameliorate renal tubular injury in a mouse model of two-hit rhabdomyolysis-related AKI. Methods: Mice were subjected to acute hemorrhagic shock and rhabdomyolysis through blood withdrawal and intramuscular glycerol injection. Mitochondria (100 μg) were freshly isolated from the soleus muscles of naïve mice. Mitochondria or placebo solution was randomly delivered into arterial catheter 1 h after the induction of AKI. The mice were euthanized 36 h later, and blood samples and kidneys were collected for analysis. Results: IVIS imaging confirmed increased expression of fluorescence-labeled allogeneic mitochondrial accumulation in AKI kidneys. Mitochondrial transplantation significantly decreased the elevated serum levels of urea nitrogen, creatinine and potassium. The protein expression of endogenous antioxidant molecules (Nrf-2, heme oxygenase-1 and superoxide dismutases) was significantly increased. The BAX-to-Bcl-2 ratio and expression of cleaved caspace-3 were also reduced in the mitochondrial-transplanted animals, indicating the attenuation of mitochondrial-mediated apoptosis in the kidney tissues of AKI mice. Histopathological examination confirmed that degrees of renal tubular injury were improved following mitochondrial transplantation. Conclusions: Transplantation of freshly isolated mitochondria augments endogenous antioxidant capacity and ameliorates mitochondrial apoptosis in the injured kidney tissues after hemorrhagic shock and rhabdomyolysis-induced AKI. Allogeneic mitochondrial transplantation could be a potential and feasible therapeutic option for prevention and management of AKI secondary to major trauma.