The mucosal epithelium is a common target of damage by chronic bacterial infections and the accompanying toxins, and most cancers originate from this tissue. We investigated whether colibactin, a potent genotoxin associated with certain strains of Escherichia coli, creates a specific DNA-damage signature in infected human colorectal cells. Notably, the genomic contexts of colibactin-induced DNA double-strand breaks were enriched for an AT-rich hexameric sequence motif, associated with distinct DNA-shape characteristics. A survey of somatic mutations at colibactin target sites of several thousand cancer genomes revealed notable enrichment of this motif in colorectal cancers. Moreover, the exact double-strand-break loci corresponded with mutational hot spots in cancer genomes, reminiscent of a trinucleotide signature previously identified in healthy colorectal epithelial cells. The present study provides evidence for the etiological role of colibactin in human cancer.