Objectives Molecular mechanism of the pathogenicity of Enterococcus faecalis (E. faecalis), a suspected endodontic pathogen, has not yet been adequately elucidated due to limited information on its virulence factors. Here we report the identification of in vivo expressed antigens of E. faecalis by using a novel immunoscreening technique called change-mediated antigen technology (CMAT) and an experimental animal model of endodontic infection. Materials and Methods Among 4,500 E. coli recombinant clones screened, 19 positive clones reacted reproducibly with hyperimmune sera obtained from rabbits immunized with E. faecalis cells isolated from an experimental endodontic infection. DNA sequences from 16 of these in vivo-induced (IVI) genes were determined. Results Identified protein antigens of E. faecalis included enzymes involved in housekeeping functions, copper resistance protein, putative outer membrane proteins, and proteins of unknown function. Conclusions In vivo expressed antigens of E. faecalis could be identified by using a novel immune-screening technique CMAT and an experimental animal model of endodontic infection. Detailed analysis of these IVI genes will lead to a better understanding of the molecular mechanisms involved in the endodontic infection of E. faecalis.