CD96 (TACTILE) is an inhibitory receptor expressed on the surface of natural killer (NK) and T-cells, where it specifically recognises nectin-like protein-5 (necl-5) on the surface of target cells. In doing so, CD96 mediates both target cell adhesion, and delivers signals to the immune cell that restrain effector function. Accordingly, CD96 is emerging as an important immune checkpoint, the modulation of which might prove beneficial in treatment of the broad range of cancers that over-express necl-5. Here, we performed a comprehensive biophysical and structural investigation into the CD96:necl-5 interaction. While the first immunoglobulin domain (D1) of CD96 bound poorly to nectin-2, this region was sufficient to mediate a strong interaction with necl-5 (KD 10.2 μM). The crystal structure of CD96 D1 bound to the entire ectodomain of necl-5 revealed that CD96 D1 adopted a V-type immunoglobulin fold that recognized necl-5 D1 via a conserved ‘lock and key’ binding topology, whilst simultaneously providing insights into the unique ligand specificity of CD96. Necl-5 recognition was underpinned by two novel structural motifs within CD96, namely an ‘ancillary key’ and an atypical ‘lock-lock’ linkage. Indeed, mutational analysis indicated that these specific interactions were critical for necl-5 binding. Overall, these data significantly broadens our understanding of the nectin receptor axis and has implications for the design of novel anti-cancer immunotherapies.