Interferon regulatory factor 6 (IRF6) is a non-canonical member of the interferon regulatory factor family of transcription factors. We recently identified IRF6 as a novel Maspin-interacting protein in mammary epithelial cells. Maspin is a tumor suppressor in the breast and has also been implicated in mammary gland morphogenesis. To explore a possible role for IRF6 in conjunction with Maspin during mammary gland growth and differentiation, we examined the expression of IRF6 and Maspin during post-utero mammary gland development using a combination of in vitro and in vivo approaches. The data revealed that the expression of IRF6 and Maspin is temporally and spatially regulated throughout mammary gland development, with maximal expression of both proteins occurring in fully differentiated, lactating lobuloalveolar cells. We further show that IRF6 adopts a lumenal localization pattern following complete epithelial cell polarization and present new evidence for the secretion of IRF6 into the milk. These results support the hypothesis that IRF6 and Maspin are important for mammary epithelial cell differentiation, and advance our understanding of the Maspin-IRF6 partnership during normal mammary gland development.