Androgen receptor (AR) plays a pivotal role in the development of primary as well as advanced castration-resistant prostate cancer. Previous work in our lab identified a novel nuclear export signal (NES) (NESAR) in AR ligand-binding domain essential for AR nucleocytoplasmic trafficking. By characterizing the localization of green fluorescence protein (GFP)-tagged NESAR, we designed and executed a yeast mutagenesis screen and isolated 7 yeast mutants that failed to display the NESAR export function. One of those mutants was identified as the splicing factor pre-mRNA processing factor 8 (Prp8). We further showed that Prp8 could regulate NESAR function using short hairpin RNA knockdown of Prp8 coupled with a rapamycin export assay in mammalian cells and knockdown of Prp8 could induce nuclear accumulation of GFP-tagged AR in PC3 cells. Prp8 expression was decreased in castration-resistant LuCaP35 xenograft tumors as compared with androgen-sensitive xenografts. Laser capture microdissection and quantitative PCR showed Prp8 mRNA levels were decreased in human prostate cancer specimens with high Gleason scores. In prostate cancer cells, coimmunoprecipitation and deletion mutagenesis revealed a physical interaction between Prp8 and AR mainly mediated by NESAR. Luciferase assay with prostate specific antigen promoter-driven reporter demonstrated that Prp8 regulated AR transcription activity in prostate cancer cells. Interestingly, Prp8 knockdown also increased polyubiquitination of endogenous AR. This may be 1 possible mechanism by which it modulates AR activity. These results show that Prp8 is a novel AR cofactor that interacts with NESAR and regulates AR function in prostate cancer cells.