About 70 % of women with advanced breast cancer (BCa) develop bone metastases. The mechanisms leading to colonization of BCa cells in the skeleton are still incompletely understood. Here, we demonstrate that mineralized extracellular matrices secreted by primary human osteoblasts (OBM) modulate cellular processes associated with BCa colonization of bone. A panel of four BCa cell lines of different bone-metastatic potential, T47D, SUM1315, MDA-MB-231 and the bone-seeking subline MDA-MB-231BO, was cultured on OBM. After 3 days, the metastatic BCa cells had undergone morphological changes on OBM and were aligned along the OBM fibers, while the non-metastatic cells showed a random orientation. Atomic Force Microscopy-based Single Cell Force Spectroscopy revealed that the metastatic cell lines adhered more strongly to OBM compared to non-metastatic cells. Function-blocking experiments indicated that b1-integrins mediated cell adhesion to OBM. Metastatic BCa cells migrated directionally and invaded OBM, which was accompanied by enhanced MMP-2 and -9 secretion. In addition, we observed gene expression changes associated with osteomimickry in BCa cultured on OBM. We further introduce a novel humanized in vivo model of bone colonization, in which polymeric electrospun tubes served as scaffolds for ectopic bone formation. Together, we have developed an experimental platform consisting of in vitro and in vivo models that will allow to further dissect the mechanisms underlying bone colonization by BCa cells.