Liposomes can improve the intracellular concentration of cytotoxic drugs, and are regarded as a possible pharmacological approach to overcome drug resistance. The kinetic analysis of subcellular drug uptake and efflux helps to elucidate the resistance mechanism which is associated with the ATP-dependent membrane transporter P-glycoprotein (P-gp). However, there are only few reports about the intracellular kinetics of liposomes. In this work, the kinetics of drug uptake and active efflux of doxorubicin (DOX) encapsulated in liposomes in both intact cells and nuclei were studied using P-gp expressing K562/DOX cells. The results show that liposomes enhanced drug accumulation in intact cells and nuclei, and improved DOX retention in nuclei after withdrawal. Furthermore, the nuclei levels of liposomal drug rose slowly and reached a plateau after 2 h incubation, whereas the free drug reached the plateau in 15 min, suggesting that it takes time for the liposomes to get from the cytoplasm to the nuclei. Our results demonstrated that liposomes not only increase DOX levels allocated to nuclei but also extended retention in the nuclei of resistant cells.