The cytolethal distending toxin (CDT) is produced by many pathogenic Gram-minus bacteria like Escherichia coli, Helicobacter hepaticus, Haemophilus ducreyi, Salmonella typhi and others. In vivo, the production of CDT by Helicobacter hepaticus induces the development of dysplastic liver nodules, thus defining CDT as a potential carcinogen. Ex vivo, CdtB, the catalytic sub-unit of CDT, is relocated into the eukaryotic cell nucleus. There, CDT induces DNA double-stranded breaks (DSBs), leading to cell cycle arrest and cell death. However, after years of study, the CDT mode of action only begins to be unrevealed and many aspects remain to be studied. To better characterize the CDT-induced DNA lesions, we are studying the biochemistry of CDT, specifically regarding its catalytic nuclease activity and relate these aspects to the overall cellular effects of the toxin. Based on the literature and on the structural homology of CdtB with the DNase I, we developed several CDT mutants for specific residues involved in the catalytic activity. The DNA binding and the nuclease activities of CdtB are studied by in vitro tests, like Supercoiled DNA cleavage and DNA binding assay. Ex vivo, CDT-induced DNA damage and the activation of specific DNA damage response (DDR) pathways have been characterized. Thanks to comet assay and immunofluorescence staining, we have shown that, depending on the CDT dose, DSBs (high doses) or SSBs (moderate doses) will be induced, the later degenerating into DSBs following the replication. In fact, after a treatment with moderate doses, CDT-induced DNA damages cause the activation of the DDR involving the RPA, ATR and CHK1 proteins, characteristic of a replicative stress. The activation of the ATM pathway, due to DSBs induction, occurs later during CDT treatment. The importance of the S-phase passage for the CDT cytotoxicity suggests that proliferating cells are more sensitive to CDT than quiescent cells. The presence of unrepaired damage can lead to cell death, whereas effective repair will allow cells to resume cell cycle. However, improper repair of DNA damage can induce genetic instability and lead to cancer. Bacterial niches containing CDT producing strains are located at epithelia that are quick renewal tissues. Understanding the effects of CDT at the cellular level is an essential step in order to understand these effects at the tissue and/or organism level as well as CDT’s involvement in bacteria pathogenicity.