Disulfide bonds form between cysteine pairs in close proximity. Disulfide bonds in plasma membrane receptors can influence structural integrity and potentially exert allosteric effects to fine-tune signalling processes. There are several conserved disulfide bonds in the extracellular domains and transmembrane helices of class B1 G protein-coupled receptors (GPCRs). These ubiquitously expressed membrane proteins are activated by peptide hormones and have a broad range of regulatory metabolic effects. We determined high-resolution cryo-EM structures of receptors of the VPAC/PAC receptor family in complex with different peptide agonists and identified a novel disulfide bond between the extracellular domain and extracellular loop 1. The relevant cysteines that formed this bond are present in some other class B1 GPCRs, but not all, suggesting the bond may also form for other, but not all receptors in this family. We analysed the structural and functional consequences of this disulfide bond using an integrated approach of cryo-EM classification and variability analysis, molecular dynamics simulations, mutagenesis and functional pharmacology assays, as well as mass spectrometry. Our data support the existence of this disulfide bond in VPAC family receptors and suggest that the disulfide can modify receptor function in a ligand-dependent manner. Furthermore, our cryo-EM data indicate that some of these disulfide bonds are transient and not ubiquitously present, at least under the conditions used for expression and purification for structure determination. In order to create realistic and engaging movies of receptor activation and conformational variability, we have loaded trajectories of atomic coordinates into Blender3D, an open-source 3D computer graphics software package. The illustrative movies based on our experimental data can both inform drug development and provide templates for communication of complex concepts to a broader, non-expert audience.