A novel androgen receptor (AR) expression system was developed in 293 human embryonic kidney cells that can mimic the biochemical activity of AR as a steroid hormone receptor (SHR) in prostate cancer (PCa) cells. This system was used to identify putative AR binding proteins in the cytosolic and nuclear compartments of the cells using a large-scale co-immunoprecipitation strategy coupled to quantitative mass spectrometry. Many previously characterized AR binding proteins were identified, which included the heat shock 70 and 90 chaperones, both regulators of SHR function. In depth bioinformatic analyses revealed that proteins involved in RNA processing, protein transport, and cytoskeletal organization were co-enriched after AR purification, suggesting a functional link between AR and these pathways in mammalian cells. For example, the endosomal and cytoskeletal proteins clathrin heavy chain and alpha-actinin-4, along with the serine-threonine protein kinase C delta (PKC delta) were specifically enriched in the nuclear compartment after AR purification. SiRNA knockdown studies and co-transcriptional reporter assays revealed that clathrin heavy chain possessed co-activator activity during AR-mediated transcription, while alpha-actinin-4 and PKC delta displayed both co-activator and co-repressor activity during AR-mediated transcription that was dependent upon their relative expression levels respectively. Lastly, immunohistochemical analyses revealed that alpha-actinin-4 levels decreased in the nucleus of high grade cancerous prostate tissues, suggesting its possible deregulation in advanced prostate cancers as previously observed in late-stage metastatic breast cancers. Taken together, these results suggest AR binds to specific protein modules in mammalian cells, and thus provides a molecular framework for interrogating AR function in normal and cancerous prostate epithelial cells.