Recent studies have shown that targeting doxorubicin to mitochondria of tumor cells can bypass the multi-drug resistance problem and inhibit tumor growth. We previously discovered that the C-9th and C-13th position-alkylated berberine derivatives possess improved mitochondria-targeting activity compared to berberine. Therefore, we hypothesize that these alkylated berberine derivatives could be utilized as potential mitochondrial-targeting ligands by inserting the alkyl chain into the liposomal bilayer membrane during the preparation of liposomes. In this research, a berberine derivate (a 16-carbon aliphatic chain was introduced to the C-9th of berberine, 9-C16 berberine) was employed to prepare mitochondria-targeting doxorubicin-loaded folic acid-conjugated polyethylene glycol(PEGylated) liposomes (MT-FOL-PLS). The results of in vitro cytotoxicity and apoptosis-inducing studies revealed that MT-FOL-PLS showed the strongest cytotoxicity and apoptosis-inducing effects in drug resistant MCF-7/adr cells in comparison with free doxorubicin and regular liposomal doxorubicin. MT-FOL-PLS enhanced cellular uptake of doxorubicin up to 15-fold compared to free doxorubicin, and targeted doxorubicin to mitochondria. In vivo and ex vivo drug distribution studies showed that MT-FOL-PLS increased the drug distribution in tumor and the administration of MT-FOL-PLS to resistant MCF-7/adr cell mouse xenografts stopped tumor growth. Our results confirmed that alkylated berberines can be exploited as mitochondrial-targeting ligands to overcome cancer multi-drug resistance, further advancing the research on active targeting of liposome delivery systems in the treatment of resistant cancer.