Yersiniosis belongs to the common foodborne diseases around the world, and frequently manifests as diarrhea that can be treated with probiotics. Colicin F Y is an antibacterial agent produced by bacteria and it is capable of specific growth inhibition of Yersinia enterocolitica, the causative agent of gastrointestinal yersiniosis. In this study, recombinant E. coli producing colicin F Y were constructed, using both known probiotic strains EcH22 and EcColinfant, and the newly isolated murine strains Ec1127 and Ec1145. All E. coli strains producing colicin F Y inhibited growth of pathogenic Y. enterocolitica during co-cultivation in vitro. In dysbiotic mice treated with streptomycin, E. coli strains producing colicin F Y inhibited progression of Y. enterocolitica infections. This growth inhibition was not observed in mice with normal gut microflora, likely due to insufficient colonization capacity of E. coli strains and/or due to spatial differences in intestinal niches. Isogenic Y. enterocolitica producing colicin F Y was constructed and shown to inhibit pathogenic Y. enterocolitica in mice with normal microflora. Evidence of in vivo antimicrobial activity of colicin F Y may have utility in the treatment of Y. enterocolitica infections.