Wingless-related MMTV integration site 5A (Wnt5a)isa noncanonical signaling WNT that is expressed in every stage of mouse mammary gland development except lactation. Using slow release pellets containing WNT5A as well as Wnt5a-null tissue, we previously showed that WNT5A acts to limit mammary development. Here, we generated transgenic mice that overexpress WNT5A in the mammary epithelium using the mouse mammary tumor virus promoter (M5a mice). Lactation was impaired in two high WNT5A-expressing lines. Lactation defects could not be explained by differences in apoptosis, lineage differentiation, milk synthesis, or secretion. Instead, misexpression of WNT5A led to a failure in oxytocin response and milk ejection. Noting the similarity between the M5a phenotype and that of mice with a mutation in connexin43 (Cx43; official gene symbol Gja1), we examined Cx43 phosphorylation and localization in M5a mice. In wild-type mice, Cx43 switched from a phosphorylated to a more hypophosphorylated form after parturition. In contrast, the phosphorylated form of Cx43 was maintained after parturition in M5a mice. Using a nontumorigenic breast cell line, MCF10A, we showed that, in addition to increasing the levels of phosphorylation of Cx43 on serine-368, ectopic expression of WNT5A reduced or blocked the amount of dye transferred between cells. In summary, we propose that WNT5A inhibits the response to oxytocin and prevents milk ejection through regulation of Cx43 function. © 2011 by the Society for the Study of Reproduction, Inc.