BACKGROUND: Several regulatory proteins are involved in Salmonella invasion. The key regulator of SPI-1 (Salmonella pathogenicity island 1 ) is hilA, a transcriptional activator encoded on SPI-1 that regulates the expression of the SPI-1 secretion system. OBJECTIVES: Importance of hilA mutation on S. enteritidis colonization and shedding in layer hens was evaluated in a long-term experiment. METHODS: Two groups of layer hens were orally inoculated with 1010 CFU of hilA and parent strains of S. enteritidis, consequently. At days 2, 7, 14, 21 and 35 post-inoculation samples were taken from cloaca and different parts of digestive and reproduction systems of euthanized birds. RESULTS: In the birds infected with parent strain, the higher numbers of colonizing bacteria in the liver, spleen, caecum, small intestine and cloaca-vagina were observed. Fecal shedding in this group was also higher than the hilA group. However, no significant differences were observed for the colonization of bacteria in magnum, isthmus and infundibulum of both groups. Using PCR method, hilA gene was only detected in tissues of parent group hens. CONCLUSIONS: This study has shown that the hilA mutant is able to colonize in internal organs; an implication of this is the possibility that genes other than hilA, or at least other mechanisms, might be involved in the invasion of S. enteritidis to the internal organs of birds.