Nectins are a family of four cell surface glycoproteins belonging to the immunoglobulin superfamily that mediate cell-cell adhesion and associated signalling pathways, thereby regulating several physiological processes including morphogenesis, growth and development of multicellular organisms. Nectins interact among themselves through their extracellular domains from the adjacent cells in both homophilic and heterophilic fashions to support cell-cell adhesion. Although nectins form homodimers as demonstrated in experimental set-ups, only the specific heterophilic interactions among nectins are physiologically relevant as shown by in vivo studies. It has been hypothesised that a conserved charged residue present at the binding interface acts as the molecular switch for heterophilic nectin-nectin recognitions. In this work, we have analysed the energetics of homophilic and heterophilic interactions of nectins, followed by surface plasmon resonance-based binding studies and complementary in silico analyses. Our findings confirm that the conserved charged residues at the binding interfaces dictate the specificity of the nectin-nectin heterophilic interactions. Furthermore, these residues also play a role in conferring higher affinity to the heterophilic interactions, thereby making them physiologically more prevalent compared to homophilic interactions. Thus, this work reveals the molecular basis of heterophilic recognitions among nectins that contribute to their physiological functions. (C) 2020 Elsevier Inc. All rights reserved.