Acquired resistance to androgen receptor (AR)-targeted therapies compels the development of novel treatment strategies for castra-tion-resistant prostate cancer (CRPC). Here, we report a profound effect of endostatin on prostate cancer cells by efficient intracellular trafficking, direct interaction with AR, reduction of nuclear AR level, and down-regulation of AR-target gene transcription. Structural modeling followed by functional analyses further revealed that phenylalanine-rich α1- helix in endostatin - which shares structural similarity with noncanonical nuclear receptor box in AR-antagonizes AR transcriptional activity by occupying the activation function (AF)-2 binding interface for coactivators and N-terminal AR AF-1. Together, our data suggest that endostatin can be recognized as an endogenous AR inhibitor that impairs receptor function through protein-protein interaction. These findings provide new insights into endostatin whose antitumor effect is not limited to inhibiting angiogenesis, but can be translated to suppressing AR-mediated disease progression in CRPC.