One major determinant of host tropism for filoviruses is viral glycoprotein (GP), which is involved in receptor binding and viral entry. Compared to Ebola GP (EGP), Marburg GP (MGP) is less well characterized in viral entry. In this study, using a human immunodeficiency virus-based pseudotyped virus as a surrogate system, we have characterized the role of MGP in viral entry. We have shown that like EGP, the mucin-like region of MGP (289-501) is not essential for virus entry. We have developed a viral entry interference assay for filoviruses, and using this assay, we have demonstrated that transfection of EGP or MGP in target cells can interfere with EGP/HIV and MGP/HIV pseudotyped virus entry in a dose-dependent manner. These results are consistent with the notion that Ebola and Marburg viruses use the same or a related host molecule(s) for viral entry. Substitutions of the non-conserved residues in MGP1 did not impair MGP-mediated viral entry. Unlike that of EGP1, individual substitutions of many conserved residues of MGP1 exerted severe defects in MGP expression, incorporation to HIV virions, and thus its ability to mediate viral entry. These results indicate that MGP is more sensitive to substitutions of the conserved residues, suggesting that MGP may fold differently from EGP.