Twenty-two atypical enteroaggregativeEscherichia coliisolates from a previous epidemiological study harboring EAEC virulence genes were examined for their adhesion properties. Nine strains showed a typical aggregative adherence (AA) pattern, while 13 strains showed variant AA, such as AA with lined up cells characteristic of the chain-like adhesion (CLA) and AA mainly to HeLa cells characteristic of the diffuse adherence (DA). The aggregative forming pilus (AFP) genesafpA2andafpRwere detected only in strain Q015B, which exhibited an AA/DA pattern. Using Tn5-based transposon mutagenesis on Q015B strain, we identified a 5517-bp open reading frame (ORF) encoding a predicted 1838-amino-acid polypeptide that is genetically related to a putative filamentous hemagglutinin identified inE. colistrain 7-233-03_S3_C2. Therefore, the ORF was namedorfHA. The regions flankingorfHAwere sequenced and two ORFs were found; upstream, an ORF that encodes a 603-amino-acid polypeptide with 99% identity to hemolysin secretion/activation proteins of the ShlB/FhaC/HecB family, and downstream, another ORF, which encodes a 632-amino-acid polypeptide with 72% identity to the glycosyltransferase EtpC. AnorfHAmutant (Q015BΔorfHA) was constructed from strain Q015B. Q015BΔorfHAstrain did not adhere to HeLa cells, whereas Q015BΔorfHAtransformed with a pACYC184 plasmid carryingorfHArestored the AA/DA phenotype of strain Q015B. Furthermore, the Q015ΔorfHAmutant had a marked effect on the ability of strain Q015B to kill the larvae ofGalleria mellonella. Our results suggest that the AA/DA pattern of strain Q015B is mediated by a hemagglutinin-associated protein which also contributes to its virulence in theG. mellonellamodel.