Autotransporters are outer membrane proteins with a complex domain structure and transmembrane topology. A C-terminal domain composed of a 12-stranded β-barrel is embedded in the outer membrane and facilitates translocation of the passenger domain to the bacterial cell surface. We discovered that paired glycine-aromatic “mortise and tenon” motifs join neighboring transmembrane β-strands; these motifs are an evolutionary-conserved feature of the entire autotransporter protein family. Mutation of the conserved glycine residue in these motifs slowed the rate of passenger domain translocation and impaired passenger domain cleavage once this domain was surface exposed. Circular dichroism, tryptophan fluorescence and thermal denaturation studies show that mutation of the mortise and tenon motif significantly perturbs barrel stability and delays completion of the folding reaction. Our results provide evidence that the β-barrel domain is directly involved in the translocation of the passenger domain to the cell surface. Furthermore, this data suggests that the high propensity glycine-aromatic residue pairings that are conserved through evolution could serve to produce modular hairpins for the folding of β-sheets, providing insight into the mechanism by which β-barrel proteins fold and insert into membranes.