Resistance to chemotherapy is a major obstacle to the successful treatment of breast cancer patients. Recently, we successfully established two vinorelbine-resistant sublines, BC-DS and BC-TS, from the human breast cancer cell line BCap37, with different 'two-stage screening methods'. Interestingly, though BC-DS and BC-TS were developed from the same BCap37 cell line with the same drug, they showed remarkable differences. Compared with the parental BCap37 cells both BC-DS and BC-TS had resistance to vinorelbine, but the resistant characterizations are both unstable. BC-DS showed increased migration capability while BC-TS showed reduced migration capability. When investigating their multidrug resistance, we found BC-DS became more sensitive to methotrexate, which suggested that combination of MTX and vinorelbine could be a new treatment strategy. Moreover, BC-DS and BC-TS overexpressed P-glycoprotein at different levels. Our research also showed that the present clinical usage of vinorelbine is reasonable. These findings suggest that the vinorelbine-induced multiple drug resistance (MDR) sublines may be used as an in vitro model not only to further elucidate possible mechanisms of MDR involved in the human breast cancer, but also to find methods to optimize the curative effect of vinorelbine in clinic.