Wnt-5a is a non-transforming Wnt protein that is implicated in cell polarity, adhesion, and motility. We have previously shown that low expression of Wnt-5a is a predictor of shorter disease-free survival in human breast cancer. Here, we investigated whether β-catenin/ E-cadherin-mediated cell-cell adhesion was affected by loss of Wnt-5a in breast carcinomas, thereby promoting a metastatic behavior of the tumor. We show that Wnt-5a stimulation of human breast epithelial cells leads to an increased Ca2+-dependent cell-cell adhesion. Furthermore, Wnt-5a/casein kinase Iα (CKIα)-specific Ser-45 phosphorylation of β-catenin is associated with an increased complex formation of β-catenin/E-cadherin. Mutation of Ser-45 decreases the β-catenin/E-cadherin association. Also, the inhibitory effect of Wnt-5a on breast epithelial cell invasion is reduced upon mutation of β-catenin-Ser-45. The Wnt-5a-CKIα-induced Ser-45 phosphorylation does not lead to degradation of β-catenin. Finally we show that human breast cancers lacking Wnt-5a protein have a significantly lower level of membrane-associated β-catenin. Down-regulation of Wnt-5a expression and subsequent reduction of membrane-associated β-catenin in invasive breast cancer, can therefore contribute to a decreased cell-cell adhesion and increased motility resulting in a higher probability for metastatic disease. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.