Widespread use of screening mammography has recently increased the detection of breast microcalcifications. These nonpalpable microcalcifications with specific features in breast tissues are clinically considered an early indicator of breast carcinoma. Our goal in this study was to develop a murine breast microcalcification model for optimizing in vivo imaging. Recombinant human BMP-2 was expressed in E. coli, and the purified bioactive protein was used as inducing factor for the production of breast microcalcifications in a murine animal model. Syngeneic breast tumors were obtained by injection of MDA-MB-231 human breast cancer cells with Matrigel into the mammary fat pad of female nude mice. Different doses of bioactive rhBMP-2 were administered either as single or multiple intraperitoneal injections or directly into tumor on a weekly basis. Three weeks after the first injection of rhBMP-2, the microcalcification of breast tumor was detected by microcomputed tomography followed by intravenous injection of radiotracer [18F] Sodium fluoride for positron emission tomography imaging. Our findings indicate that rhBMP-2 induced microcalcifications of breast tumor by both systemic and direct injection of rhBMP-2 into tumors in a dose-dependent manner. Although little is known about the molecular mechanism of microcalcification, here we report a new murine model of human breast tumor induced microcalcification by rhBMP-2 to optimize in vivo imaging methods and to study the role of BMP-2 as a mediator of pathological mineralization and bone-like microcalcification formation in breast tumor. This BMP-2-induced microcalcification model may allow us to discriminate the type of microcalcification in tumors and to perform quantitative analysis on the calcification as a new detection strategy for early identification of pathological mineralization of breast tissues in women.