Autotransporters (ATs) are outer membrane proteins (OMPs) that contain both a channel-forming, β-barrel transmembrane domain (β-domain) and a passenger domain, which is the secreted functional moiety. AT β-domains form a 12-stranded β-barrel in the outer membrane that facilitates translocation of the passenger domain onto the bacterial cell surface. Following translocation, some AT passenger domains remain tethered to their β-domains, whereas the passenger domains of others are cleaved autoproteolytically from their β-barrels and secreted into the extracellular milieu. Glycine is the most abundant internal amino acid in naturally occurring β-barrel membrane proteins, including AT β-barrels. While glycine is an intrinsically destabilizing residue in β-sheets, this destabilization can be ‘rescued’ by specific cross-strand pairing of glycines with aromatic residues such that the aromatic side-chain in the adjacent antiparallel strand adopts an unusual and unfavorable position or rotamer to bend over the glycine residue, shielding the hydrogen-bonded backbone from solvent. Certainly, glycine-tyrosine is the most favorable motif for backbone hydrogen bond interactions. In this study, we used Pet from Escherichia coli as a model AT of the self-cleaving type, to investigate aromatic rescue of glycines in β-sheets in vivo, for the first time. Here, we focused on the pairing of Gly58 and Tyr85 in β-strand three and four of the Pet β-domain (Petβ), demonstrating that both of these residues are highly conserved in the β-domain of ATs from a variety of bacterial species. Furthermore, independent substitutions of Gly58 and Tyr85 to alanine affected the stability of Petβ in the bacterial outer membrane, results consistent with the high-propensity of glycine-tyrosine cross-strand pairs involved in aromatic rescue in β-barrel proteins. Future studies will aim to determine if this favourable pairing of residues is important for in vivo folding and stability of β-barrel proteins in general.