Molecular interactions mediated by engagement of the Herpes virus entry mediator (HVEM) with members of TNF and Ig superfamily generate distinct signals in T cell activation pathways that modulate inflammatory and inhibitory responses. HVEM interacts with CD160 and B and T lymphocyte attenuator (BTLA), both members of the immunoglobulin (Ig) superfamily, which share a common binding site that is unique from that of LIGHT, a TNF ligand. BTLA or CD160 engagement with HVEM deliver inhibitory or stimulatory signals to the host immune response in a context dependent fashion, whereas HVEM engagement with LIGHT results in pro-inflammatory responses. We identified a mutation in human HVEM, G89F, which directly interferes with the human LIGHT interaction, but interestingly, also differentially modulates the binding of human BTLA and CD160 via an apparent allosteric mechanism involving recognition surfaces remote from the site of the mutation. Specifically, the G89F mutation enhances binding of CD160, while decreasing that of BTLA to HVEM in cell-based assays. Molecular dynamics simulations for wild-type and G89F mutant HVEM, bound to different sets of ligands, were performed to define the molecular basis of this unexpected allosteric effect. These results were leveraged to design additional human HVEM mutants with altered binding specificities.