Contaminated poultry and egg products are major sources of Salmonella enterica serovar Enteritidis (S. enteritidis, SE) infections in humans. Colonization of SE in chicken reproductive tract results in the production of contaminated commercial shell-eggs and fertilized hatchery eggs. The complex pathogen-host interactions during SE colonization of chicken reproductive tract are largely unknown. This study was aimed at determining the pathogenic roles of the type three secretion systems (TTSS-1 and TTSS-2) in SE infection of chicken oviduct epithelial cells (COEC). A series of SE strains carrying mutations in the genes encoding structure or effector proteins of TTSS-1 and TTSS-2 were constructed. The invasiveness and intracellular survival rate of each SE strain as well as the host innate immune responses induced by the infections were evaluated. The results demonstrate that both TTSS-1 and TTSS-2 are required by SE to invade COEC which involve genes encoding effector proteins SipA, SopB, SopE2, and PipB. In addition to their involvement in host cell invasion, sipA and sipB are also necessary for the survival or replication of SE inside COEC. Inactivation of TTSS-2 genes (ssaV and pipB) resulted in an enhanced bacterial proliferation inside COEC. The data from this study also show that SE infection triggers pro-inflammatory responses in COEC and TTSS-1 is involved in the expression of iNOS and IL-8, a CXC chemokine. TTSS-1 and TTSS-2 are not necessary for induction of K203, MIP-1β, and IL-10 or suppression of TGF-β3 in COEC.