Mitochondrial damage is a critical pathological factor in various forms of tissue injury, and specific therapies with high biosafety are desirable. Inspired by the natural role of extracellular vesicles (EVs) in regulating mitochondrial metabolism, we report that healthy tissue-derived mitochondria-rich EVs (Ti-mitoEVs) can boost mitochondrial biogenesis for regenerative medicine. Ti-mitoEVs that contain abundant functional mitochondria can be highly efficiently isolated from muscles via an optimized method. In vitro, Ti-mitoEV treatment increased mitochondrial biogenesis and reduced mitochondrial damage in recipient cells, and these effects occurred at least partly via mitochondrial genome transfer. In vivo, Ti-mitoEV treatment attenuated diverse types of tissue injury (e.g., muscle and kidney) by rescuing mitochondrial injury and its associated inflammation. As natural nanovesicles, the therapeutic potency of mitoEVs can be further improved by integrating them with other engineering methods. This study highlights the promising role of Ti-mitoEVs in boosting mitochondrial biogenesis, positioning them as potential therapies for treating various types of tissue injury characterized by mitochondrial damage.