Various species of the intestinal microbiota have been associated with the development of colorectal cancer(1,2), but it has not been demonstrated that bacteria have a direct role in the occurrence of oncogenic mutations. Escherichia coli can carry the pathogenicity island pks, which encodes a set of enzymes that synthesize colibactin(3). This compound is believed to alkylate DNA on adenine residues(4,5) and induces double-strand breaks in cultured cells(3). Here we expose human intestinal organoids to genotoxic pks(+)E. coli by repeated luminal injection over five months. Whole-genome sequencing of clonal organoids before and after this exposure revealed a distinct mutational signature that was absent from organoids injected with isogenic pks-mutant bacteria. The same mutational signature was detected in a subset of 5,876 human cancer genomes from two independent cohorts, predominantly in colorectal cancer. Our study describes a distinct mutational signature in colorectal cancer and implies that the underlying mutational process results directly from past exposure to bacteria carrying the colibactin-producing pks pathogenicity island. Organoids derived from human intestinal cells that are co-cultured with bacteria carrying the genotoxic pks(+) island develop a distinct mutational signature associated with colorectal cancer.