Extensive tissue remodeling occurs in breast tissue during pregnancy, resulting in growth and development of the mammary gland associated with extensive vascular remodeling, which is thought to be dependent on vascular endothelial growth factor (VEGF). We show here that the endogenous antiangiogenic splice isoform of VEGF, VEGF(165)b, is normally expressed in nonlactating human and mouse breast, and is down-regulated in WT mice during lactation. To demonstrate the physiological role of VEGF(165)b in mammary tissue, we generated transgenic (TG) mice expressing VEGF(165)b, under the control of the mouse mammary tumor virus (MMTV) enhancer/promoter. These mice increase expression of VEGF(165)b in mammary tissue during mammary development. The offspring of TG mothers, but not TG fathers, die shortly after birth. The female TG mice have fewer blood vessels, less blood in the mammary tissue, and impaired alveolar coverage of the fat pad, and do not produce sufficient milk for nourishment of their pups. These findings demonstrate that endogenous overexpression of VEGF(165)b in the mammary gland inhibits physiological angiogenesis and that the regulation of the balance of VEGF isoforms is a requirement for mammary alveolar development and milk production. This study provides the first evidence for the role of endogenous antiangiogenic VEGF isoforms in normal physiology--their down-regulation is required for effective milk production.