Many persistent and chronic bacterial infections are associated with the formation of large cell aggregates and biofilms that are difficult to treat. One mechanism employed by bacteria to aggregate and form biofilms involves the expression of self-associating surface-located autotransporter proteins (AT). AT proteins represent the largest group of outer membrane and secreted proteins in Gram-negative bacteria. Despite their abundance and role in bacterial pathogenesis, most AT proteins have not been structurally characterized and there is a paucity of detailed information with regard to their mode of action. Here we present the crystal structure of the functional passenger domain of Ag43, a prototypic self-associating AT protein from uropathogenic Escherichia coli.  We demonstrate that its unique L-shaped structure drives the formation of cell aggregates via a molecular Velcro-like handshake mechanism. This work provides new insight into the structure-function mechanisms that facilitate bacterial interactions during infection.