Ebola from the filoviridae family of viruses causes severe and mostly fatal hemorrhagic fevers in primates and has been listed as a category IV pathogen by the NIH. Viral Protein 40 (VP40), the major matrix protein of Ebola virus, regulates the assembly and budding of the virus and alone harbors the ability to form virus-like particles (VLPs) from human cells. We hypothesize that VP40 is a high affinity lipid binding and membrane curvature-inducing protein with specificity for plasma membrane (PM) lipids. This specificity leads to localization of VP40 to the PS-rich inner leaflet of the PM and formation of VLPs. Using fluorescence spectroscopy to investigate VP40 binding and insertion within lipid vesicles (POPC:POPS) containing brominated lipids. Because the fluorescence of the tryptophan is variably quenched depending on its distance from the bromine atoms on the lipid acyl chain, a tryptophan introduced into the membrane binding interface was utilized as a probe to detect the depth of membrane penetration of VP40. Results were indicative of VP40's high affinity and specificity for PS in a PS-concentration dependent manner demonstrating the robust ability of VP40 to penetrate membranes. Further analysis of VP40 membrane insertion revealed a depth of penetration more than halfway into one monolayer of the membrane. Data also confirmed that VP40 binds with nanomolar affinity to vesicles that recapitulated the PM in comparison to the nuclear membrane. In addition VP40-mutants, which inhibit membrane binding and penetration, obstruct VLP formation and release. Cellular assays confirmed the lipid-binding specificity of VP40 in the PM of different cell lines and also demonstrated that deep membrane penetration is essential for VLP. We predict that these results will elucidate the molecular basis of VP40 induced membrane curvature changes, a prerequisite to the PM deformation required for VLP production.