Normal development of the mammary gland is fundamental for lactation and requires exquisite cellular coordination. The gap junction proteins Cx26, Cx32 and Cx30 have been reported in luminal epithelial cells of the mammary gland while Cx43 is expressed in myoepithelial cells and in the stroma. This project aimed to determine the role of Cx43 in mammary gland development and function by employing a mouse model (Gja1(Jrt/+)) that harbors an autosomal dominant Cx43 mutation, and wild-type (WT) littermates. The highly phosphorylated species of Cx43, the total gap junction plaque number and gap junctional intercellular communication were reduced in mammary glands from Gja1(Jrt/+) mice, resulting in delayed development of the ducts. At parturition, Cx43 levels and gap junction plaque numbers were still reduced in Gja1(Jrt/+) mice, yet the morphology of the gland did not differ from WT mice. Interestingly, while higher than WT levels of beta-casein and WAP were present in the gland of Gja1(Jrt/+) mice, oxytocin failed to stimulate milk delivery to the ducts. Together, these data revealed that decreased levels of Cx43 result in delayed development of the mammary gland and a full complement of Cx43 is necessary for the expulsion of milk.