The type VI secretion system (T6SS) is a virulence factor for many Gram-negative bacteria. Salmonella genus harbors five phylogenetically distinct T6SS loci encoded in Salmonella Pathogenicity Islands (SPIs) SPI-6, SPI-19, SPI-20, SPI-21 and SPI-22, which are differentially distributed among serotypes. The T6SSs encoded in SPI-6 and SPI-19 contribute to pathogenesis of serotypes Typhimurium and Gallinarum in mice and chickens, respectively. Salmonella Dublin is a pathogen restricted to cattle where it causes a systemic disease. Also, it can colonize other hosts such as chickens and mice, which can act as reservoirs of this serotype. Salmonella Dublin harbors the genes for both T6SSSPI₋₆ and T6SSSPI₋₁₉. This study has determined the contribution of T6SSSPI₋₆ and T6SSSPI₋₁₉ to host-colonization by Salmonella Dublin using avian and murine models of infection. Competitive index experiments showed that, a mutant strain lacking both T6SSs (∆T6SSSPI₋₆/∆T6SSSPI₋₁₉) presents a strong colonization defect in cecum of chickens, similar to the defect observed for the ∆T6SSSPI₋₆ mutant, suggesting that this serotype requires a functional T6SSSPI₋₆ for efficient colonization of the avian gastrointestinal tract. Colonization of mice was also defective, although to a lesser extent than in chickens. In contrast, the T6SSSPI₋₁₉ was not necessary for colonization of either chickens or mice. Transfer of T6SSSPI₋₆, but not T6SSSPI₋₁₉, restored the ability of the double mutant to colonize both animal hosts. Our data indicate that Salmonella Dublin requires only the T6SSSPI₋₆ for efficient colonization of mice and chickens, and that the T6SSSPI₋₆ and T6SSSPI₋₁₉ are not functionally redundant.