During the 2013-2016 Ebola virus (EBOV) epidemic in Western Africa, an A82V mutation emerged in the envelope glycoprotein (GP) that persisted in most circulating isolates. Previous studies demonstrated that A82V increased GP-mediated membrane fusion and altered its dependence on host factors. The mechanistic basis for these observations, in particular the impact of A82V on the conformational changes in GP that are needed for membrane fusion, has not been evaluated in molecular detail. Here, using molecular dynamics simulations, fluorescence correlation spectroscopy, and single-molecule Förster resonance energy transfer imaging, we specify the molecular mechanism by which A82V alters GP conformation to enhance viral entry. In so doing, we identify an allosteric network of interactions that links the receptor-binding site to the fusion loop of GP. Thus, the naturally occurring A82V mutation can tune the conformational dynamics of EBOV GP to enhance fusion loop mobility and subsequent viral fusion and infectivity in human cells.