SalmonellaPathogenicity Island 19 (SPI-19) encoded type VI secretion system (T6SS) is a virulence factor present in few serotypes of S. enterica, including S. Dublin, S. Gallinarum and S. Pullorum. Comparative genomic sequence analysis revealed that the gene clusters of SPI-19 showed high homology to T6SS2 locus from avian pathogenic Escherichia coli, implying the similar T6SS locus is potentially related to the host adaption of both pathogens. Deletion of SPI-19 in S. Pullorum caused the dramatically decreased invasion into chicken LMH epithelial cells and HD-11 macrophages, and affected survival of Salmonella within both cells. In addition, deletion of SPI-19 caused the decreased colonization of S. Pullorum in chicken liver, spleen, ileum, and cecum at the initial infection stage, and induced rapid bacterial clearance. However, the SPI-19/T6SS had no effect on bacterial killing activity and induction of cytotoxicity to HD-11 macrophages. Further analysis demonstrated SPI-19/T6SS was involved in mediating the inhibition of host Th1 and Th2 immune responses, resulting in persistent colonization of S. Pullorum in hosts.