Prolactin receptor activation contributes to the progression and motility of human breast cancer. This event activates multimeric signaling pathways, including the activation of the Vav family of guanine nucleotide exchange factors. To detect novel proteins interacting with Vav, yeast two-hybrid analysis was performed and demonstrated an interaction between the serine/threonine NIMA-related family kinase p56^Nek3 and Vav1. The prolactin-dependent interaction of Nek3 with Vav1 and Vav2, was confirmed by co-immunoprecipitation analysis. Prolactin stimulation of T47D cells induced Nek3 kinase activity, and the interaction of Vav2/Nek3 with the prolactin receptor. Increased Nek3 levels upregulated Vav2 serine and tyrosine phosphorylation, while knock-down of Nek3 resulted in a reduction of Vav2 phosphorylation. Activation of GTPase Rac-1 in CHO transfectants, required both Nek3 and Vav2, and was inhibited by the over-expression of a kinase inactivating Nek3 mutant. However, overexpression of either Nek3 or kinase-inactive Nek3 had no effect on Vav2-potentiated Stat5-mediated gene expression. Overexpression of kinase inactive Nek3 in T47D cells led to a 50% increase in apoptosis vs. controls. These data suggest that the prolactin-mediated activation of Nek3 contributes differentially to Vav2 signaling pathways involving Rac1 and Stat5 and implicates Nek3 during prolactin-mediated actions in breast cancer.