Abstract: Objective:To prepare perillyl alcohol (POH)-loaded distearoyl phosphoethanolamine (DSPE)-polyethylene glycol (PEG)-(3- carboxy propyl) triphenyl phosph- onium bromide (TPP)-liposomes (POH/DPT-LN), and to investigate the physicochemical characteristics, mitochondrial targeting and the inhibitory effect on breast cancer MDA-MB-231 cells. Methods:POH/DPT-LN was prepared by thin film dispersion method. Cell counting kit-8 (CCK-8) assay, cell scratch experiment, Transwell, apoptosis experiment, JC- 1 staining experiment and western blotting were used to evaluate the mitochondrial targeting and tumor-inhibitory effects of drug-loading systems. Results:POH/DPT-LN was uniform and round, with an average particle size of (141.0±0.8) nm, zeta potential of (29.5±2.8) mV, PDI of (0.185±0.006), encapsulation efficiency and drug loading capacity of (88.6± 1.8)% and (17.7±0.3)%, respectively. The IC50 of POH/DPT-LN was 63.32 µg/mL, and it had a good inhibitory effect on the growth of MDA-MB-231 cells. Compared with the POH monotherapy group, the migration ability of MDA-MB-231 cells in the POH/DPT-LN group was significantly weakened, and the effect on inhibiting cell invasion was obvious (P＜0.01). POH/DPT-LN group increased the accumulation concentration of POH in the mitochondria and enhanced the cell killing effect (P＜0.01). POH/DPT-LN can significantly increase the protein expression of pro-apoptotic P53, Bax, Caspase-3 and reduce the protein expression of anti-apoptotic Bcl-2. Conclusion:POH/DPT-LN has good encapsulation efficiency and drug loading capacity. It can target POH drugs into mitochondria, more effectively inhibit the proliferation of MDA-MB-231 breast cancer cells, and enhance the anti-tumor effect of drugs