Toxin-antitoxin systems are prevalent in different bacterial organisms and are encoded in the chromosomal or plasmid DNA. The primary function of a plasmid toxin-antitoxin module is to stabilize the plasmid by eliminating plasmid-free daughter cells through a post segregation killing mechanism. In Campylobacter jejuni, a pathogen that causes human gastroenteritis, multiple plasmids have been identified, but there have not been any reports on toxin-antitoxin systems. In this study, a toxin-antitoxin system is identified and characterized in the pVir plasmid of C. jejuni IA 3902. pVir encodes a type IV secretion system and is found to be involved in natural transformation and virulence in Campylobacter. Comparative genomic analysis of the pVir45 and pVir46 genes in C. jejuni indicates that they encode a putative toxin-antitoxin system that belongs to the RelE/StbE family. Cloning and expression of the pVir46 gene alone in Escherichia coli inhibited bacterial growth, but co-expression of pVir45-46 led to the restoration of growth, confirming the functions deduced from comparative genomic analysis. The plasmid stability assay in C. jejuni showed that the toxin-antitoxin system is necessary for maintaining the stability of pVir because deletion of the pVir46 gene resulted in loss of the plasmid during passage in conventional media. qRT-PCR data demonstrated that expression of the pVir45-46 genes varied with growth phase, as early logarithmic phase had the highest level of gene expression. Together these results establish that pVir45-46 encode a functional toxin-antitoxin system in C. jejuni, which is required for ensuring the stability of the pVir plasmid. The stable maintenance of pVir may be necessary for optimal virulence of C. jejuni IA 3902.