Reversal of cancer drug resistance remains a critical challenge in chemotherapy. Mitochondria-targeted drug delivery has been suggested to mitigate drug resistance in cancer. To overcome the intrinsic limitations in conventional mitochondrial targeting strategies, we develop mitochondrial temperature-responsive drug delivery to reverse doxorubicin (DOX) resistance in lung cancer. Results demonstrate that the thermoresponsive nanocarrier can prevent DOX efflux and facilitate DOX accumulation and mitochondrial targeting in DOX-resistant tumors. As a consequence, thermoresponsive nanocarrier enhances the cytotoxicity of DOX and reverses the drug resistance in tumor-bearing mice. This work represents the first example of mitochondrial temperature-responsive drug delivery for reversing cancer drug resistance.