Mitochondrial-oriented delivery of anticancer drugs has been considered as a promising strategy to improve the antitumor efficiency of chemotherapeutics. However, the physiological and biological barriers from the injection site to the final mitochondrial action site remain great challenges. Herein, a novel mitochondrial-targeted multifunctional nanocomplex based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers (MPC) is designed to enhance drug accumulation in mitochondria. MPC possesses various functions such as extracellular pH response, superior cellular uptake, lysosomal escape, and mitochondrial targeting. In detail, MPC was formed by two oppositely charged HPMA copolymers, that is, positively charged mitochondrial-targeting guanidine group-modified copolymers and charge-reversible 2,3-dimethylmaleic anhydride (DMA)-modified copolymers (P-DMA). It was validated that MPC could remain stable in the blood circulation (pH 7.4) but could be cleaved to expose the positive charge of the guanidine group immediately in response to the mild acidity of tumor tissues (pH 6.5). The gradual exposure of positively charged guanidine will simultaneously facilitate endocytosis, endosomal/lysosomal escape, and mitochondrial targeting. The in vitro experiments showed that compared with copolymers without guanidine modification, the cellular uptake and mitochondrial-targeting ability of MPC in the simulated tumor environment (MPC@pH6.5) separately increased 4.3- and 23.8-fold, respectively. The in vivo experiments were processed on B16F10 tumor-bearing C57 mice, and MPC showed the highest accumulation in the tumor site and a peak tumor inhibition rate of 82.9%. In conclusion, multifunctional mitochondrial-targeting HPMA copolymers provide a novel and versatile approach for cancer therapy.