Background and Purpose: Androgen receptor (AR), a ligand-activated transcription factor, is a master regulator in the development and progress of prostate cancer (PCa). A major challenge for the clinically used AR antagonists is the rapid emergence of resistance induced by some point mutations in the AR ligand binding domain (LBD), and therefore discovery of novel anti-AR therapeutics that can combat mutation-induced resistance is quite demanding. Therein, blocking the interaction between AR and DNA represents an innovative strategy to overcome resistance of traditional antagonists towards the AR LBD. Experimental Approach: In this study, an integrated docking-based virtual screening (VS) strategy based on the crystal structure of the DNA binding domain (DBD) of AR was conducted to search for novel AR antagonists, and then a series of bioassays including bio-layer interferometry (BLI) and RNA-seq were used to evaluate the biological activities of these compounds. Key Results: Among the tested compounds, 2-(2-butyl-1,3-dioxoisoindoline-5-carbox-amido)-4,5-dimeth-oxybenzoicacid (Cpd39) was identified as a potential hit, which was competent to block the binding of the AR DBD to androgen receptor response (ARE) and showed decent potency against AR transcriptional activity. Furthermore, Cpd39 was capable of effectively inhibiting the proliferation of PCa cell lines (i.e., LNCaP, PC3, DU145, and 22RV1) and reducing the expression of not only the full-length AR but also the splice variant AR-V7. Conclusion and Implications: The novel AR DBD-ARE blocker Cpd39 could serve as a starting point for the development of new therapeutics for castration-resistant PCa.