Autotransporters are a large family of bacterial outer membrane proteins that play key roles in the pathogenesis of many infectious diseases. The virulence function of autotransporters depends on their correct assembly at the bacterial cell surface. Assembly requires the C-terminal b-barrel domain to be folded and inserted into the outer membrane to facilitate translocation of the N-terminal passenger domain to the bacterial cell surface, where it exerts its virulence function after being cleaved from its b-barrel only to remain attached to this domain in a non-covalent manner. The autotransporter Antigen 43 (Ag43) is found in many types of pathogenic Escherichia coli where it functions in adhesion and biofilm formation by mediating cell-cell aggregation. How the Ag43 passenger domain folds into its biologically active conformation is unknown. Using biochemical and biophysical methods, our current study showed that the fifth extracellular loop of the Ag43 b-barrel domain, which connects b-strands 9 and 10, is required for the efficient folding and function of the Ag43 passenger domain. The refolding of Ag43 in vitro also provided evidence that cleavage of the Ag43 passenger domain occurs via an autocatalytic mechanism. These findings provide valuable insights into the assembly pathway of Ag43 and reveal new potential therapeutical targets in treating bacterial infections.