Avian pathogenic Escherichia coli (APEC) strains frequently cause extraintestinal infections and are responsible for significant economic losses in the poultry industry worldwide. APEC isolates are closely related to human extraintestinal pathogenic E. coli (ExPEC) strains and may also act as pathogens for humans. Known APEC virulence factors include adhesins such as type 1 fimbriae and curli, iron acquisition systems, and cytotoxins. Here we show that APEC strain SEPT362, isolated from a septicemic hen, expresses a type VI secretion system (T6SS); causes cytoskeleton rearrangements; and invades epithelial cells, replicates within macrophages, and causes lethal disease in chicks. To assess the contribution of the T6SS to SEPT362 pathogenesis, we generated two mutants, hcp (which encodes a protein suggested to be both secreted and a structural component of the T6SS) and clpV (encoding the T6SS ATPase). Both mutants showed decreased adherence and actin rearrangement on epithelial cells. However, only the hcp mutant presented a mild decrease in its ability to invade epithelial cells, and none of these mutants were defective for intramacrophage replication. Transcriptome studies showed that the level of expression of type 1 fimbriae was decreased in these mutants, which may account for the diminished adhesion and invasion of epithelial cells. The T6SS seems to be important for the disease process, given that both mutants were attenuated for infection in chicks. These results suggest that the T6SS influences the expression of type 1 fimbriae and contributes to APEC pathogenesis.Extraintestinal pathogenic Escherichia coli (ExPEC) strains cause a wide range of diseases, including urinary tract infections, newborn meningitis, abdominal sepsis, and septicemia (59). ExPEC pathotype subgroups include avian pathogenic E. coli (APEC), uropathogenic E. coli (UPEC), neonatal meningitis E. coli (NMEC), and septicemic E. coli (61). In poultry, APEC strains are a frequent cause of extraintestinal infections leading to respiratory or systemic disease. Colisepticemia, the most severe systemic disease, is characterized by pericarditis, perihepatitis, airsacculitis, synovitis, and peritonitis and is responsible for significant economic losses in the poultry industry worldwide (34). APEC strains are closely related to human ExPEC strains, with significant similarity existing between UPEC and APEC (46, 47). Additionally, it has been suggested that some closely related clones can be involved in extraintestinal infections in humans and poultry, suggesting no host specificity for these types of isolates (27,43,46,47,58).The pathogenicity mechanisms of APEC are varied and remain largely uncharacterized. Established APEC virulence traits include iron uptake systems, the production of cytotoxins, and adhesins (40,41,76). Adhesins play an important role in APEC pathogenesis, and the best-characterized adhesins include type 1, P, and curli fimbriae and temperature-sensitive hemagglutinin (Tsh). These adhesins are required for the full virulenc...