Pannexin1 (Panx1) is an important transmembrane protein which mediates cellular communication through formation of oligomeric (heptamer) channels.[1] However, the undesired opening of these channels is known to be related to a wide range of pathologies (including liver and cardiovascular inflammatory diseases) and therefore, Panx1 channel blockade may represent an innovative strategy for the treatment of these diseases.[2] Recent cryogenic-electron microscopy (Cryo-EM) structures of Panx1 channels[3] have highlighted that a particular helical sequence found in the extracellular domain of this protein assembly overlaps strikingly with a well-known peptide inhibitor, called 10Panx1.[4] Following a rational design, a series of macrocyclic ("stapled") 10Panx1 mimetic peptides were synthesized to pinpoint where the side chain-to-side chain triazole-based crosslinks needed to be introduced for optimal inhibition. In a next step, to further improve these promising, peptide-based Panx1 channel inhibitors, the molecular flexibility was reduced by introducing "rigid motifs" within the macrocycles. The combination of global constraints via macrocyclization of the peptide backbone and local constraints by use of custom amino acids brought again improvements in both in vitro channel inhibition, but also proteolytic stability. The obtained analogues largely outperform the existing, non-selective inhibitors described to date. Altogether, the results showcase this promising approach, and indicate yet again that side chain conformation is key to molecular recognition.