Filoviruses are the causative agents of an increasing number of disease outbreaks in human populations, including the current unprecedented Ebola virus disease EVD outbreak in Western Africa. One obstacle to controlling these epidemics is our poor understanding of the host range of filoviruses and their natural reservoirs. Here, we investigated the role of the intracellular filovirus receptor, Niemann-Pick C1 NPC1 as a molecular determinant of Ebola virus EBOV host range at the cellular level. Whereas human cells can be infected by EBOV, a cell line derived from a Russells viper Daboia russellii VH-2 is resistant to infection in an NPC1-dependent manner. We found that VH-2 cells are resistant to EBOV infection because the Russells viper NPC1 orthologues bound poorly to the EBOV spike glycoprotein GP. Analysis of panels of viper-human NPC1 chimeras and point mutants allowed us to identify a single amino acid residue in NPC1, at position 503, that directionally influenced both its binding to EBOV GP as well as its viral receptor activity in cells. Significantly, this single residue change perturbed neither NPC1s endosomal localization nor its housekeeping role in cellular cholesterol trafficking. Together with other recent work, these findings identify sequences in NPC1 that are important for viral receptor activity by virtue of their direct interaction with EBOV GP, and suggest that they may influence filovirus host range in nature. Broader surveys of NPC1 orthologues from vertebrates may delineate additional sequence polymorphisms in this gene that control susceptibility to filovirus infection.