OBJECTIVE The effect of breast cancer cells on the normal function of osteoblasts remains unclear. This study was to investigate the effect of conditioned medium from two types of human breast cancer lines on osteoblastic proliferation, differentiation and mineralization. METHODS Fetal rat carvarial cells were treated with conditioned medium from MCF-7 estrogen-receptor(ER) positive, or MDA-MB-231 ER negative, breast cancer cells. Proliferation was measured by MTT assay, alkaline phosphatase (ALP) activity was assessed by the p-nitrophenyl phosphate (PNPP) method, and mineralized nodules were confirmed by alizarin red S (ARS) staining and the area of bone nodules was measured to observe the mineralization capacity. RESULTS When osteoblasts were treated with conditioned medium from MDA-MB-231 or MCF-7, the cells became long and spindle-like, assumed a fibroblastic morphology. Conditioned medium from breast cancer cells inhibited proliferation and differentiation of osteoblasts, compared with those cultured with vehicle control medium. The proliferation inhibition rates of culture with 50% conditioned medium from MDA-MB-231 and MCF-7 cells on osteoblasts were 18.1%, 13.0%, 19.2%, 19.3% and 15.8%, 20.8%, 33.9%, 28.7% on day 1, day 3, day 5, and day 7, respectively (P<0.01). Moreover, incubation with conditioned medium inhibited ALP activity and bone-nodule formation of osteoblasts in a dose-dependent manner. Addition of 50% conditioned medium from MDA-MB-231 and MCF-7 cells inhibited ALP activity by 31.9% and 47.5% (P<0.01), and diminished the area of bone nodules by 89% and 74%, respectively, compared with the vehicle control group. CONCLUSIONS Breast cancer cells inhibit proliferation, decrease ALP activity and disrupt the mineralization process of osteoblasts.