The Dengue virus (DENV) prM protein consists of two moieties, the pr and M domains. Apart from preventing the premature fusion activity of the DENV E protein, prM has several other unknown biological roles, displaying both protein-protein and membrane-protein interactions. Although the prM protein is an essential component of the DENV viral cycle, little is known about its biological functions and what regions of this protein are responsible for said functions. By performing an exhaustive study of membrane rupture induced by a prM peptide library on simple and complex model membranes as well as their ability to modulate the phospholipid phase transitions of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine and 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-glycerol], we identified six membranotropic regions on the prM protein. Apart from the previously identified two transmembrane segments of the protein, one of these regions probably interacts with the fusion E protein and another one, the stem segment, would interact with the membrane modulating its structure. These data will help us understand the molecular mechanism of viral entry and morphogenesis, allow the identification of new targets for the treatment of Dengue virus infection, and make possible the future development of DENV entry inhibitors.