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Pyrvinium was identified as the first small molecule inhibitor of the androgen receptor (AR) DNA-binding domain (DBD). It was also among the first small molecules shown to directly inhibit the activity of AR splice variants (ARVs), which has important clinical implications in the treatment of castration-resistant prostate cancer. Important questions about pyrvinium's mechanism of action remain. Here, we demonstrate through mutational analysis that amino acids 609 and 612 are important for pyrvinium action. Nuclear magnetic resonance demonstrates a specific interaction between a soluble pyrvinium derivative and the AR DBD homodimer-DNA complex. Chromatin immunoprecipitation and electrophoretic mobility shift assay experiments demonstrate that, despite an interaction with this complex, pyrvinium does not alter the DNA-binding kinetics in either assay. AR immunoprecipitation followed by mass spectrometry was used to identify proteins whose interaction with AR is altered by pyrvinium. Several splicing factors, including DDX17, had reduced interactions with AR in the presence of pyrvinium. RNA sequencing of prostate cancer cells treated with pyrvinium demonstrated changes in splicing, as well as in several other pathways. However, pyrvinium did not alter the levels of ARVs in several prostate cancer cell lines. Taken together, our new data pinpoint the direct interaction between pyrvinium and AR DBD and shed light on the mechanism by which it inhibits AR transcriptional activity.