Yersinia enterocolitica employs a type III secretion system (TTSS) to target virulence factors (e.g. YopE) into the cytosol of the host cells. We utilized the TTSS to introduce a recombinant antigen directly into the cytosol of host cells and to investigate the potential of Y. enterocolitica and Y. pseudotuberculosis as live carrier for vaccines. The model antigen ovalbumin (Ova) was fused to defined secretion or translocation domains of the Yersinia effector protein YopE and introduced into attenuated mutant strains of Y. enterocolitica and Y. pseudotuberculosis. In vitro experiments showed secretion and translocation of YopE-Ova hybrid proteins into host cells. To investigate the resulting immune responses, mice expressing transgenic Ova-specific T cell receptors were used. Both Y. enterocolitica and Y. pseudotuberculosis mutants induced efficaciously Ova-specific CD8+ T cell responses. The translocation domain of YopE was required for induction of CD8+ T cell responses in vivo, but not for T cell responses induced in vitro. The in vivo frequency of Ova-specific splenic T cells was up to six-fold higher in mice immunized with YopE-Ova-translocating Y. enterocolitica/Y. pseudotuberculosis mutants than in control mice. The Ova-specific T cells were shown to produce high amounts of IFN-gamma. We did not observe significant Ova-specific CD4+ T cell or antibody responses upon vaccination with either of the strains. In conclusion, Yersinia live carrier vaccine strains are suitable to target antigens into the MHC class I pathway and stimulate CD8+ T cell responses and thus, might be useful in vaccine approaches against intracellular pathogens.