2-Amino-3-methylimidazo[4,5- f ]quinoline (IQ) is a genotoxic/carcinogenic compound formed in meat and fish during cooking. Following absorption in the upper part of the gastrointestinal tract, IQ is mainly metabolized in the liver by xenobiotic-metabolizing enzymes. Among them, UDP-glucuronosyl transferases lead to harmless glucuronidated derivatives that are partly excreted via the bile into the digestive lumen, where they come into contact with the resident microbiota. The purpose of this study is to investigate if microbial β-glucuronidase could contribute to IQ genotoxicity by releasing reactive intermediates from IQ glucuronides. We constructed a β-glucuronidase-deficient isogenic mutant from a wild-type Escherichia coli strain carrying the gene uidA encoding this enzyme and compared the genotoxicity of IQ in gnotobiotic rats monoassociated with the wild-type or the mutant strain. The Comet assay performed on colonocytes and hepatocytes showed that the presence of β-glucuronidase in the digestive lumen dramatically increased (3-fold) the genotoxicity of IQ in the colon. This deleterious effect was paralleled by slight modifications of the pharmacokinetics of IQ. The urinary and faecal excretion of the parent compound and its conjugated derivatives reached a maximum 24–48 h after gavage in rats harbouring the β-glucuronidase-deficient strain. In rats associated with the wild-type strain, the kinetics of urinary excretion showed a biphasic curve with a second, smaller peak after 144 h. This is the first in vivo demonstration that bacterial β-glucuronidase plays a pivotal role in the genotoxicity of a common food-borne carcinogen.