The forkhead box transcription factor FOXC1 plays a critical role in embryogenesis and the development of many organs. Its mutations and high expression are associated with many human diseases including breast cancer. Although FOXC1 knockout mouse studies showed that it is not required for mammary gland development during puberty, it is not clear whether its overexpression alters normal mammary development in vivo. To address this question, we generated transgenic mice with mammary-specific FOXC1 overexpression. We report that transgenic FOXC1 overexpression suppresses lobuloalveologenesis and lactation in mice. This phenotype is associated with higher percentages of estrogen receptor-, progesterone receptor-, or ki67-positive mammary epithelial cells in the transgenic mice at the lactation stage. We also show that expression of the Elf5 transcription factor, a master regulator of mammary alveologenesis and luminal cell differentiation, is markedly reduced in mammary epithelial cells of transgenic mice. Likewise, levels of activated Stat5, another inducer of alveolar expansion and a known mediator of the Elf5 effect, are also lowered in those cells. In contrast, the cytokeratin 8-positive mammary cell population with progenitor properties is elevated in the transgenic mice at the lactation stage, suggesting inhibition of mammary cell differentiation. These results may implicate FOXC1 as a new important regulator of mammary gland development.