The androgen receptor (AR) plays an essential role in the development of prostate cancer, and androgen-deprivation therapy is used as a first-line treatment for prostate cancer. However, under androgen-deprivation therapy, castration-resistant prostate cancer inevitably arises, suggesting that the interacting transcriptional coregulators of AR are promising targets for developing novel therapeutics. In this study, we used novel proteomic techniques to evaluate the AR interactome, including biochemically labile binding proteins, which might go undetected by conventional purification methods. Using rapid immunoprecipitation mass spectrometry of endogenous proteins, we identified enhanced at puberty 1 (EAP1) as a novel AR coregulator, whereas its interaction with AR could not be detected under standard biochemical conditions. EAP1 enhanced the transcriptional activity of AR via the E3 ubiquitin ligase activity, and its ubiquitination substrate proteins included AR and HDAC1. Furthermore, in prostate cancer specimens, EAP1 expression was significantly correlated with AR expression as well as a poor prognosis of prostate cancer. Together, these results suggest that EAP1 is a novel AR coregulator that promotes AR activity and potentially plays a role in prostate cancer progression.