Marburg virus (MARV), which belongs to the Filoviridae family of viruses, causes hemorrhagic fever in humans and non-human primates that is often fatal. MARV is a lipid-enveloped virus that during the replication process extracts its lipid coat from the plasma membrane of the host cell it infects. MARV encodes seven genes, one of which is its matrix protein VP40 (mVP40), which regulates assembly and budding of the virions. To date, a dearth of information is available on mVP40 lipid binding properties. Here we have investigated the in vitro and cellular mechanisms by which mVP40 associates with lipid membranes. mVP40 associates with anionic membranes in a nonspecific manner that was dependent upon the anionic charge density of the membrane. These results are consistent with recent structural determination of mVP40, which elucidated an mVP40 dimer with a flat and extensive cationic lipid-binding interface.