The prolactin receptor (PrlR) is a member of the cytokine receptor superfamily that lacks an intrinsic kinase domain and relies on the cytoplasmic Jak tyrosine kinases to transduce signals. Prolactin-induced Jak2 activation and consequent tyrosine phosphorylation of the receptor and downstream signaling molecules have been studied, but phosphorylation of the PrlR on serine or threonine residues has not been reported. Here we describe a novel interaction between the PrlR and the phosphoserine/phosphothreonine-binding 14-3-3 proteins. This association is mediated by the KCST391WP motif, which occurs in the major functional isoform of the human receptor and is conserved among a wide variety of species. Mutagenesis of threonine 391 to alanine significantly impaired 14-3-3 binding to the PrlR in both glutathione S-transferase pulldown and coimmunoprecipitation assays. In breast carcinoma and mouse mammary epithelial cell lines, the endogenous receptor was found to associate with glutathione S-transferase-14-3-3 proteins independent of prolactin stimulation. A phospho-specific peptide antibody was generated and used to demonstrate phosphorylation of Thr391 in vivo. Phosphorylation of this site was found to be sensitive to okadaic acid, a specific inhibitor of serine/threonine protein phosphatases. Interestingly, the T391A PrlR mutant exhibited increased basal and prolactin-induced tyrosine phosphorylation compared with the wild-type receptor. This was accompanied by a ligand-induced increase in protein kinase B and Erk activation but not that of Stat5a. Phosphorylation of the receptor on Thr391 may therefore provide a new mechanism by which prolactin signaling is attenuated.