Treatment of advanced prostate cancer with androgen deprivation therapy inevitably renders the tumors castration-resistant and incurable. Under these conditions, neuroendocrine differentiation of prostate cancer (CaP) cells is often detected and neuropeptides released by these cells may facilitate the development of androgen independence. Exemplified by gastrin-releasing peptide (GRP), these neuropeptides transmit their signals through G protein-coupled receptors, which are often overexpressed in prostate cancer, and aberrantly activate androgen receptor (AR) in the absence of androgen. We developed an autocrine neuropeptide model by overexpressing GRP in LNCaP cells and the resultant cell line, LNCaP-GRP, exhibited androgen-independent growth with enhanced motility in vitro. When orthotopically implanted in castrated nude mice, LNCaP-GRP produced aggressive tumors, which express GRP, prostate-specific antigen, and nuclear-localized AR. Chromatin immunoprecipitation studies of LNCaP-GRP clones suggest that GRP activates and recruits AR to the cognate promoter in the absence of androgen. A Src family kinase (SFK) inhibitor, AZD0530, inhibits androgen-independent growth and migration of the GRP-expressing cell lines, and blocks the nuclear translocation of AR, indicating the involvement of SFK in the aberrant activation of AR and demonstrating the potential use of SFK inhibitor in the treatment of castration-resistant CaP. In vivo studies have shown that AZD0530 profoundly inhibits tumor metastasis in severe combined immunodeficient mice implanted with GRP-autocrine LNCaP cells. This xenograft model shows autocrine, neuropeptide- and Src kinase-mediated progression of androgen-independent CaP postcastration, and is potentially useful for testing novel therapeutic agents.