Constitutively active androgen receptor splice variants (AR-V) lacking the ligand-binding domain have been implicated in the pathogenesis of castration-resistant prostate cancer and in mediating resistance to newer drugs that target the androgen axis. AR-V regulates expression of both canonical AR targets and a unique set of cancer-specific targets that are enriched for cell-cycle functions. However, little is known about how AR-V controls gene expression. Here, we report that two major AR-Vs, termed AR-V7 and AR^v567es, not only homodimerize and heterodimerize with each other but also heterodimerize with full-length androgen receptor (AR-FL) in an androgen-independent manner. We found that heterodimerization of AR-V and AR-FL was mediated by N- and C-terminal interactions and by the DNA-binding domain of each molecule, whereas AR-V homodimerization was mediated only by DNA-binding domain interactions. Notably, AR-V dimerization was required to transactivate target genes and to confer castration-resistant cell growth. Our results clarify the mechanism by which AR-Vs mediate gene regulation and provide a pivotal pathway for rational drug design to disrupt AR-V signaling as a rational strategy for the effective treatment of advanced prostate cancer. Cancer Res; 75(17); 3663–71. ©2015 AACR.