Abstract The androgen receptor (AR) is a hormone-activated transcription factor implicated in the development and progression of prostate cancer. The AR contains an N-terminal domain (NTD), followed by DNA binding (DBD) and ligand-binding (LBD) domains. Upon binding of androgens to the LBD, the AR moves into the nucleus where it interacts with target genes via the conserved AR-DBD. Prostate cancer treatment involves the use of small-molecules to block androgen production or to compete with androgens for binding to the AR-LBD. Drug-resistance occurs when LBD mutations render small-molecule treatment ineffective or when constitutively active AR splice variants, lacking the LBD, become overexpressed. We recently identified a surface pocket on the AR-DBD that may serve as an alternative drug-target site. The DBD-specific, small molecule compounds presented here block transcriptional activity of full-length and splice variant AR forms across multiple cell lines and show no cross-reactivity with related steroid nuclear receptors. The inhibition is lost when residues in the binding pocket are mutated or when the AR-DBD is replaced with a different DNA binding domain. Biophysical techniques were used to explore the interaction of these compounds with the purified, recombinant DBD protein. Gene expression analysis demonstrated the down-regulation of AR target genes following compound treatment, without any obvious expression signature associated with toxicity. The compounds affected DNA binding in vitro and also blocked interactions of the AR with chromatin inside the nucleus. Finally, the lead compound suppressed the growth of LNCaP tumor xenografts and was accompanied by down-regulation of AR target genes, such as PSA. These results suggest a mechanism whereby our developed inhibitory compounds interfere with DNA binding by the AR, potentially leading to effective treatment of advanced, castration-resistant prostate cancers. Citation Format: Kush Dalal, Mani Roshan-Moniri, Aishwariya Sharma, Huifang Li, Fuqiang Ban, Mohamed D. Hassona, Michael Hsing, Kriti Singh, Eric LeBlanc, Scott Dehm, Emma Tomlinson Guns, Artem Cherkasov, Paul S. Rennie. Selectively targeting the DNA-binding domain of the androgen receptor as a prospective therapy for prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1778. doi:10.1158/1538-7445.AM2015-1778