Prolactin (PRL) 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 (never in mitosis A)-related family kinase p56Nek3 and Vav1. The PRL-dependent interaction of Nek3 with Vav1 and Vav2 was confirmed by coimmunoprecipitation analysis. PRL stimulation of T47D cells induced Nek3 kinase activity and the interaction of Vav2/Nek3 with the PRL receptor. Increased Nek3 levels up-regulated Vav2 serine and tyrosine phosphorylation, whereas knockdown of Nek3 resulted in a reduction of Vav2 phosphorylation. Activation of guanosine triphosphatase Rac-1 in Chinese hamster ovary transfectants required both Nek3 and Vav2 and was inhibited by the overexpression of a kinase inactivating Nek3 mutant. However, overexpression of either Nek3 or kinase-inactive Nek3 had no effect on Vav2-potentiated signal transducer and activator of transcription 5-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 PRL-mediated activation of Nek3 contributes differentially to Vav2 signaling pathways involving Rac1 and signal transducer and activator of transcription 5 and implicates Nek3 during PRL-mediated actions in breast cancer.