Autotransporter (AT) proteins are a core component of the Type V secretion system, the most prevalent secretion system in Gram-negative bacteria. AT proteins share several common features: (i) an N-terminal leader sequence, (ii) a passenger domain which determines the protein’s functional characteristics, and (iii) a C-terminal translocation or β-barrel domain which is embedded in the outer membrane. AT proteins are grouped into four subclasses: Type Va, Vb, Vc and Vd. However, recent studies have revealed a new Type Ve subclass, which are similar to Type Va AT proteins, but are reversed in that they possess an N-terminal translocation domain and a C-terminal passenger domain. In this study, our aim was to identify and characterize Escherichia coli Type Ve AT proteins. 83 complete E. coli genomes available on the NCBI database were first analyzed to identify genes encoding for these proteins. Several genes were identified, and further analysis showed that the genes encoded for proteins which separated into several distinct groups. The strain SMS-3-5 possessed six different type Ve AT proteins representing each of these groups, and hence was selected for further study. A prevalence screen using both in silico methods to probe complete E. coli genomes and a PCR screen to study the E. coli Reference (ECOR) strain collection showed that several of the genes were more common than others. The genes were cloned into a plasmid expression vector, and a segment representing the passenger domain of each protein, respectively, was purified and used to generate a specific polyclonal antibody. Four proteins were found to be expressed on the surface of recombinant strains via immunofluorescence microscopy. These same four proteins were also able to mediate biofilm formation on abiotic surfaces when overexpressed in recombinant strains. Further characterization of the E. coli type Ve AT proteins is still underway.