Human Respiratory Syncytial virus (RSV) is the leading cause of infantile bronchiolitis in the developed world and of childhood deaths in resource-poor settings. The elderly and the immunosuppressed are also affected. It is a major unmet target for vaccines and anti-viral drugs. RSV assembles and buds from the host cell plasma membrane by forming infections viral particles which are mostly filamentous. A key interaction during RSV assembly is the interaction of plasma membrane lipids with the Matrix (M) protein layer forming at assembly sites. Although the structure of RSV M protein dimer is known, it is unclear how the viral M proteins interact with certain plasma membrane lipids to promote viral assembly. Here, we demonstrate that M proteins cluster at the plasma membrane by selectively binding with phosphatidylserine (PS). Our in vitro studies suggest that M binds PS lipid as dimers and M mutant with a phosphomimetic substitution inhibits interaction with PS lipids. The presence of other negatively charged lipids like PI(4, 5)P2 does not affect RSV M ability to bind, while cholesterol negatively affects M interaction with lipids. Moreover, we show that the initial .