The prostate transglutaminase, TGase-4, is a member of the transglutaminase family and is uniquely expressed in the prostate gland. The function of the protein is largely unknown, although an influence on cell motility and adhesion has been indicated. The present study investigated the impact of the differential expression of TGase-4 in human prostate cancer cells on RON, the hepatocyte growth factor-like/macrophage-stimulating protein (HGF-L/MSP) receptor, mediated cellular functions. Using human prostate cancer cell lines and prostate tissues, we demonstrated that human TGase-4 had a high degree of co-localisation with RON, primarily at the cell periphery and cell-cell adhesion region. High levels of TGase-4 expression in CAHPV10 cells and in PC3 cells engineered to over-express TGase-4 were associated with significantly increased cell motility in response to HGF-L, a clear contrast to wild-type and control cells. Neutralising antibody to RON and rhHGFL/MSP had no further bearing on the increased motility in TGase-4 over-expressing cells, although they had profound effect on the control cells. Akt pathway inhibitor significantly diminished the effect induced by HGF-L in the cells. Finally, over-expression of TGase-4 in prostate cancer cells resulted in autophosphorylation of RON. It is concluded that TGase-4 expression is intrinsically linked to the activation of RON in prostate cancer cells and that this autoactivation of RON contributes to the increased cell motility in TGase-4 expressing cells.