Epimorphin is known as a mesenchymal factor involved in epithelial morphogenesis. This protein has, however, a curious nature in that only certain of its molecules are transported to extracellular regions after having undergone complex conformational changes. In the present study, we generated an N-terminally modified recombinant epimorphin fragment as a substitute for the extracellular epimorphin and examined in detail how this polypeptide affects cellular behavior in a model cell system. As immunohistochemical studies revealed that epimorphin is abundant in regions close to endothelial cells in venulae, we chose endothelial cells as the model cell and investigated the influence of this polypeptide on their cellular behaviorin vitro.The recombinant epimorphin guided the endothelial cells to align themselves in tandem and to present a branched morphology in the three-dimensional culture system. We also discovered that the endothelial cells were induced to secrete several cytokines, including those involved in angiogenesis, and were suppressed in terms of proliferation by this molecule. These results suggest that epimorphin has a regulatory role in the activation of endothelial cells and is active in supporting the resulting cellular arrangement.