The human microbiota is suggested to be a reservoir of antibiotic resistance (ABR) genes, which are exchangeable between transient colonizers and residing bacteria. In this study, the transfer of ABR genes from Enterococcus faecalis to Listeria monocytogenes and to commensal bacteria of the human gut microbiota was demonstrated in a colonic fermentation model. In the first fermentation, an E. faecalis donor harboring the marked 50-kb conjugative plasmid pRE25* and a chromosomal marker was co-immobilized with L. monocytogenes and infant feces. In this complex environment, the transfer of pRE25* to L. monocytogenes was observed. In a second fermentation, only the E. faecalis donor and feces were co-immobilized. Enumeration of pRE25* and the donor strain by quantitative PCR revealed an increasing ratio of pRE25* to the donor throughout the 16-day fermentation, indicating the transfer of pRE25*. An Enterococcus avium transconjugant was isolated, demonstrating that ABR gene transfer to gut commensals occurred. Moreover, pRE25* was still functional in both the E. avium and the L. monocytogenes transconjugant and transmittable to other genera in filter mating experiments. Our study reveals that the transfer of a multiresistance plasmid to commensal bacteria in the presence of competing fecal microbiota occurs in a colonic model, suggesting that commensal bacteria contribute to the increasing prevalence of antibiotic-resistant bacteria.