Identifying cellular factors that determine susceptibility to infection can help us understand how Ebola virus is transmitted. We asked if the EBOV receptor Niemann-Pick C1 (NPC1) could explain why reptiles are resistant to EBOV infection. We demonstrate that cells derived from the Russell’s viper are not susceptible to infection because EBOV cannot bind to viper NPC1. This resistance to infection can be mapped to a single amino acid residue in viper NPC1 that renders it unable to bind to EBOV GP. The newly solved structure of EBOV GP bound to NPC1 confirms our findings, revealing that this residue dips into the GP receptor-binding pocket and is therefore critical to the binding interface. Consequently, this otherwise well-conserved residue in vertebrate species influences the ability of reptilian NPC1 proteins to bind to EBOV GP, thereby affecting viral host range in reptilian cells.