To determine the effect of the down-regulation of osteopontin (OPN) expression on the sensitivity of breast cancer cells to doxorubicin, a chemotherapeutic drug, OPN shRNA was used to transfect the OPN-positive breast cancer cell line MDA-MB-231. The expression of OPN mRNA and protein was analyzed by RT-PCR and Western blotting, respectively. The cell cycle distribution and apoptosis were analyzed by flow cytometry. Transfected MDA-MB-231 cells were treated with different concentrations of doxorubicin. Growth of MDA-MB-231 cells and IC50 of doxorubicin were determined by MTT assay. Expression of OPN mRNA and OPN protein of cells transfected with OPN shRNA was significantly decreased when compared to the negative controls (P<0.05). The number of MDA-MB-231 cells in the S phase was significantly increased, from 22.77 to 43.67%. Transfection increased apoptosis from 3.61 to 4.91%. Furthermore, the proliferation of MDA-MB-231 cells transfected with OPN shRNA was significantly decreased when compared to the negative control (transfected with empty vector). Sensitivity of MDA-MB-231 cells to doxorubicin was enhanced after treatment with OPN shRNA. The IC50 values of doxorubicin of the OPN-transfected group, the MDA-MB-231 cells and the negative transfected group were 0.13588, 0.83869 and 0.79652 µg/ml, respectively. Down-regulation of OPN significantly inhibits expression of OPN protein in MDA-MB-231 breast cancer cells, reduces cell proliferation and increases their sensitivity to doxorubicin.