The AP-1 transcription factor is activated by multiple growth factors that are critical regulators of breast cell proliferation. We previously demonstrated that AP-1 blockade inhibits breast cancer cell growth in vitro. Yet a specific role of AP-1 in normal mammary gland development has not been studied. Using a bi-transgenic mouse expressing an inducible AP-1 inhibitor (Tam67), we found that the AP-1 factor regulates postnatal proliferation of mammary epithelial cells. Mammary epithelial proliferation was significantly reduced after AP-1 blockade in adult, prepubertal, pubertal, and hormone-stimulated mammary glands. In pubertal mice, mammary cell proliferation was greatly reduced, and the cells that did proliferate failed to express Tam67. We also observed structural changes such as suppressed branching and budding, reduced gland tree size, and less fat pad occupancy in developing mammary glands after AP-1 blockade. We further demonstrated that Tam67 suppressed the expression of AP-1-dependent genes (TIMP-1, vimentin, Fra-1, and fibronectin) and the AP-1-dependent growth regulatory genes (cyclin D1 and c-myc) in AP-1-blocked mammary glands. We therefore conclude that AP-1 factor is a pivotal regulator of postnatal mammary gland growth and development.