Prostate transglutaminase (TGase-4 or TGaseP) is an enzyme that is uniquely expressed in prostate tissues. The function of the TGase, implicated in the cell-matrix, is yet to be fully established. In the present study, we investigated the role of TGase-4 in tumor-endothelial cell interactions, by creating a panel of prostate cancer cell lines that have different expression profiles of human TGase-4. Here, we report that prostate cancer cells PC-3, when over-expressing TGase-4 (PC-3(TGase4exp)) increased their ability to adhere to quiescent and activated (by hepatocyte growth factor) endothelial cells. In contrast, the prostate cancer cell CAHPV-10, which expressed high levels of TGase-4, reduced the adhesiveness to the endothelial cells after TGase-4 expression was knocked down. By using frequency based electric cell impedance sensing, we found that TGase-4 mediated adhesion resulted in a change in impedance at low frequency (400 Hz), indicating a paracellular pathway disruption. The study further showed that expression of TGase-4 rendered the cells to exert regulation of endothelial interaction by bypassing the ROCK pathway. It is therefore concluded, that TGase-4 plays a pivotal role in the interaction between endothelial cells and prostate cancer cells, an action which is independent of the ROCK pathway.