Evidence is accumulating that a growing number of bacterial toxins act by modulating the eukaryotic cell cycle machinery. In this context, we provide evidence that a protein toxin named cytotoxic necrotizing factor 1 (CNF1) from uropathogenic Escherichia coli is able to block cell cycle G(2)/M transition in the uroepithelial cell line T24. CNF1 permanently activates the small GTP-binding proteins of the Rho family that, beside controlling the actin cytoskeleton organization, also play a pivotal role in a large number of other cellular processes, including cell cycle regulation. The results reported here show that CNF1 is able to induce the accumulation of cells in the G(2)/M phase by sequestering cyclin B1 in the cytoplasm and down-regulating its expression. The possible role played by the Rho GTPases in the toxin-induced cell cycle deregulation has been investigated and discussed. The activity of CNF1 on cell cycle progression can offer a novel view of E. coli pathogenicity.