Various species of the intestinal microbiota have been associated with the development of colorectal cancer (CRC), yet a direct role of bacteria in the occurrence of oncogenic mutations has not been established. Escherichia coli can carry the pathogenicity island pks, which encodes a set of enzymes that synthesize colibactin. This compound is believed to alkylate DNA on adenine residues and induces double-strand breaks in cultured cells. Here, we expose human intestinal organoids to genotoxic pks E. coli by repeated luminal injection over a period of 5 months. Whole-genome sequencing of clonal organoids before and after this exposure reveals a distinct mutational signature, absent from organoids injected with isogenic pks-mutant bacteria. The same mutational signature is detected in a subset of 5,876 human cancer genomes from two independent cohorts, predominantly in CRC. Our study describes a distinct mutational signature in CRC and implies that the underlying mutational process directly results from past exposure to bacteria carrying the colibactin-producing pks pathogenicity island.