The Cytolethal Distending Toxin (CDT) is a genotoxin produced by several pathogenic bacteria. It is generally admitted that CDT induces double-strand breaks (DSB) and cell cycle arrest in G2/M-phase, in an ATM-dependent manner. Most of these results were obtained at high dose (over 1 μg ml−1) of CDT and late after treatment (8–24 h). We provide here evidence that the Escherichia coli CDT (EcCDT) – at low dose (50 pg ml−1 or LD50) and early after treatment (3–6 h) – progressively induces DNA DSB, mostly in S-phase. DSB formation is related to the single-strand breaks induction by CDT, converted into DSB during the S-phase. We also show that homologous recombination is mobilized to these S-phase-associated DSB. This model unveils a new mechanism for CDT genotoxicity that may play a role in cells partly deficient in homologous recombination.