Atovaquone, an FDA-approved drug for malaria, is known to inhibit mitochondrial electron transport. In this study, the synthesized mitochondria-targeted atovaquone (Mito-ATO) increased mitochondrial uptake in both cancer cells and immunosuppressive cells in the TIME. Local intratumoral injection of Mito-ATO into primary tumors triggered potent T cell immune responses locally and in distant tumor sites.