In the present study we demonstrate that leupaxin mRNA is overexpressed in prostate cancer (PCa) as compared to normal prostate tissue by using cDNA arrays and quantitative RT-PCR analyses. Moderate to strong expression of leupaxin protein was detected in approximately 22% of the prostate cancer tissue sections analyzed and leupaxin expression intensities were found to be significantly correlated with Gleason patterns/scores. In addition, different leupaxin expression levels were observed in PCa cell lines and at the subcellular level leupaxin was usually localized in focal adhesion sites. Furthermore, mutational analysis and transfection experiments of LNCaP cells using different GFP-leupaxin constructs demonstrated that leupaxin contains functional nuclear export signals in its LD3 and LD4 motifs, thus shuttling between the cytoplasm and the nucleus. We could also demonstrate for the first time that leupaxin interacts with the androgen receptor in a ligand-dependent manner and serves as a transcriptional activator of this hormone receptor in PCa cells. Down-regulation of leupaxin expression using RNAi in LNCaP cells resulted in a high rate of morphological changes, detachment, spontaneous apoptosis and a reduction of PSA secretion. In contrast, knockdown of leupaxin expression in androgen-independent PC-3 and DU 145 induced a significant decrease of both the invasive capacity and motility. Our results therefore indicate that leupaxin could serve as a potential progression marker for a subset of prostate cancers and may represent a novel co-activator of the androgen receptor. Leupaxin could function as a putative target for therapeutic interventions of a subset of advanced prostate cancers.