The human and animal pathogen Campylobacter fetus is divided into the two subspecies C. fetus subsp. fetus and C. fetus subsp. venerealis. Despite their high conservation on the genome level, they display different host and niche preferences, which may be linked to a small number of unique genes in each subspecies. However, the current knowledge about C. fetus pathogenicity is restricted due to the lack of genetic tools, and therefore new tools for manipulating C. fetus genetically may broaden our understanding of the pathogens virulence mechanisms. A method for generating site-specific gene disruptions in bacteria is the insertion of group II introns from Targetron plasmids, by a mechanism called retrohoming. In this study, the original Targetron plasmid was modified for application in C. fetus. C. fetus subsp. venerealis strain 84-112 harbors fic (filamentation induced by cyclic AMP) genes, located chromosomally or on the extra-chromosomal element ICE 84-112. Recently, it was unveiled that fic genes, consisting of a Fido- and/or an inhibitory motif, form functional toxin-antitoxin modules in C. fetus. To unravel further interactions between predicted toxins and antitoxins, co-expression studies were implemented in E. coli, and this study focuses on the Fti (Fic toxin inhibitor) proteins, which harbor a predicted inhibitory motif and are extra-chromosomally located. A crosstalk between chromosomally- and extra-chromosomally located fic/fti was indicated, as extra-chromosomal Fti3 was shown to abrogate toxicity of the chromosomally encoded Fic2 toxin. The importance of Fti proteins in C. fetus was underlined by the finding that Fti4 ensures normal cell survival of fic4-expressing cells despite disruption of Fic4s inhibitory motif. This study extends the knowledge about the recently identified toxin-antitoxin systems in C. fetus.