Receptors for vasoactive intestinal peptide (VIP) and the human epidermal growth factor family of tyrosine kinase receptors (HER) are potent promoters of cell proliferation, survival, migration, adhesion and differentiation in prostate cancer cell lines. In this study, we analyzed the cross-talk between both classes of receptors through the regulation of HER2 transactivation and expression by VIP. Three growth-hormone-releasing hormone analogs endowed with antagonistic activity for VIP receptors (JV-1-51, -52, and -53) abrogated the autocrine/paracrine stimuli of VIP on androgen-independent PC3 cells in the absence or the presence of 10% fetal bovine serum. Semiquantitative and real-time quantitative RT-PCR together with Western blotting showed increased expression levels of both mRNA and proteins for HER2 and HER3 in PC3 and androgen-dependent LNCaP prostate cancer cells as compared to non-neoplastic RWPE-1 cells. VIP (100 nM) stimulated the expression levels of both HER2 and HER3 in PC3 cells in a time-dependent manner. Whereas these effects were relatively slow, VIP rapidly (0.5 min) increased HER2 tyrosine phosphorylation. This pattern of HER transactivation was blocked by H89, a protein kinase A (PKA) inhibitor, as well as by the specific VIP antagonist JV-1-53, indicating the involvement of VIP receptors and PKA activity in phosphorylated HER2 formation. These findings support the merit of further studies on the potential usefulness of VIP receptor antagonists and both HER2 antibodies and tyrosine kinase inhibitors for prostate cancer therapy.