Multidrug resistance is the most predominant phenomenon leading to chemotherapy treatment failure in breast cancer patients. Despite many studies having suggested that overexpression of epidermal growth factor receptor (EGFR) is a potent predictor of malignancy in cancers, systematic research of EGFR in multidrug resistant (MDR) breast cancer cells is lacking. In order to clarify the role of EGFR in MDR breast cancer cells, MCF7/Adr expressing relatively higher EGFR, and its parental cell line MCF7 expressing relatively lower EGFR, were chosen for this study. Knockdown of EGFR by siRNA in MCF7/Adr cells showed that EGFR siRNA inhibits cell migration, invasion and proliferation in vitro; converse effects were observed in MCF7 cells transfected with pcDNA3.0-EGFR plasmid. Moreover, we found that EGFR upregulated migration and invasion via EMMPRIN, MMP2 and MMP9 in addition to promoting cell cycle passage via elevation of cyclin D1 and CDK4 in MDR breast cancer cells. Interestingly, MCF7/Adr cells not expressing EGFR showed significant decrease of P-glycoprotein (P-gp) and ABCG2 expression levels, and became more sensitive to treatment of adriamycin (ADR) and paclitaxel (Taxol); the above results indicated that MDR of cancer cells is related to S-phase arrest. In conclusion, EGFR is an important factor enhancing the malignancy of MDR breast cancer cells, partially, inducing MDR. Anti-EGFR therapy may improve outcome in chemorefractory breast cancer patients.