IGFs (IGF-I and IGF-II) are essential for development, and their bioactivities are tightly regulated by six related IGF-binding proteins (IGFBPs). IGFBP-5 is the most highly conserved binding protein and is expressed in several key developmental lineages as well as in multiple adult tissues including the mammary gland. To explore IGFBP-5 actions in vivo, we produced IGFBP-5 knockout (KO) mice. Whole-body growth, selected organ weights, and body composition were essentially normal in IGFBP-5 KO mice, presumably because of substantial compensation by remaining IGFBP family members. The IGFBP-5 KO mice also exhibited normal mammary gland development and were capable of nursing their pups. We then directly evaluated the proposed role of IGFBP-5 in apoptosis and remodeling of mammary gland during involution. We found that the process of involution after forced weaning was delayed in IGFBP-5 KO mice, with both the appearance of apoptotic cells and the reappearance of adipocytes retarded in mutant mice, compared with controls. We also determined the effects of IGFBP-5 deletion on mammary gland development in pubertal females after ovariectomy and stimulation with estradiol/progesterone. In this paradigm, IGFBP-5 KO mammary glands exhibited enhanced alveolar bud formation consistent with enhanced IGF-I action. These results demonstrate that IGFBP-5, although not essential for normal growth, is required for normal mammary gland involution and can regulate mammary gland morphogenesis in response to hormone stimulation.