Avian pathogenic is the causative agent of extra-intestinal infections in birds known as colibacillosis, which can manifest as localized or systemic infections. The disease affects all stages of poultry production, resulting in economic losses that occur due to morbidity, carcass condemnation and increased mortality of the birds. APEC strains have a diverse virulence trait repertoire, which includes virulence factors involved in adherence to and invasion of the host cells, serum resistance factors, and toxins. However, the pathogenesis of APEC infections remains to be fully elucidated. The Type 6 secretion (T6SS) system has recently gained attention due to its role in the infection process and protection of bacteria from host defenses in human and animal pathogens. Previous work has shown that T6SS components are involved in the adherence to and invasion of host cells, as well as in the formation of biofilm, and intramacrophage bacterial replication. Here, we analyzed the frequency of T6SS genes , , , and in a collection of APEC strains and their potential role in virulence-associated phenotypes of APECO18. The T6SS genes were found to be significantly more prevalent in APEC than in fecal isolates from healthy birds. Expression of T6SS genes was analyzed in culture media and upon contact with host cells. Mutants were generated for , , , and and characterized for their impact on virulence-associated phenotypes, including adherence to and invasion of host model cells, and resistance to predation by Deletion of the aforementioned genes did not significantly affect adherence and invasion capabilities of APECO18. Deletion of reduced resistance of APECO18 to predation by , suggesting that T6SS is involved in the virulence of APECO18.