Cytolethal distending toxins (CDTs) are secreted by Gram-negative pathogens as both soluble proteins and associated with extracellular vesicles (BEVs). CDT production by the enteric pathogen Campylobacter jejuni has been associated with colorectal tumorigenesis. Despite CDT being a major virulence factor for C. jejuni, little is known about this toxin. To address this question, we generated C. jejuni cdtA, cdtB and cdtC mutants by double homologous recombination using suicide vectors harbouring a promoter-driven kanamycin resistance (KmR) cassette. C. jejuni cdtA and cdtC mutant bacteria expressed wildtype (wt) levels of cdtB/C and cdtA/B, respectively, whereas the cdtB mutant expressed reduced levels of cdtA/C. Immunoblotting revealed that C. jejuni cdtA bacteria and BEVs did not contain CdtB/C subunits. Similar results were observed for a C. jejuni cdtA mutant constructed using a promoterless KmR cassette. C. jejuni cdtB and cdtC bacteria and BEVs only contained CdtA. The loss of Cdt subunit production in the mutants was attributed to transcriptional and post-transcriptional modifications resulting from the mutagenesis procedure. Nanoparticle tracking analyses showed that C. jejuni wildtype (wt) and cdt mutants released similar numbers of BEVs with comparable morphologies and size distributions. As reported previously for C. jejuni bacteria, we found that the BEVs isolated from wt but not cdt mutants induced cell cycle arrest at the G2 phase in HCT-116 epithelial cells. “Surface shearing” with proteinase K and surface plasmon resonance showed that the majority of Cdt subunits are located inside BEVs, with small quantities detected on the surface. In conclusion, we have performed the first detailed characterisation of CDT release in C. jejuni BEVs. We suggest that BEV-associated CDT may be a new and important factor in C. jejuni gastroenteritis and associated diseases.