The enterohepatic recycling of a drug consists of its biliary excretion and intestinal reabsorption, which is sometimes accompanied by hepatic conjugation and intestinal deconjugation reactions. β-Glucuronidase, an intestinal bacteria-produced enzyme, can break the bond between a biliary excreted drug and glucuronic acid. Antibiotics such as ciprofloxacin can reduce the enterohepatic recycling of glucuronide-conjugated drugs. In this study, we established an in vitro system to evaluate the β-glucuronidase-mediated deconjugation of the irinotecan metabolite SN-38-G to its active SN-38 form and the effect of ciprofloxacin thereon. SN-38 formation increased in a time-dependent manner from 5 to 30 min. in the presence of β-glucuronidase. Ciprofloxacin and phenolphthalein-β-d-glucuronide (PhePG), a typical β-glucuronidase substrate, significantly decreased SN-38-G deconjugation and, hence SN-38 formation. Similarly, the antibiotics enoxacin and gatifloxacin significantly inhibited the conversion of SN-38-G to SN-38, which was not observed for levofloxacin, streptomycin, ampicillin and amoxicillin/clavulanate. Ciprofloxacin showed a dose-dependent inhibitory effect on the β-glucuronidase-mediated conversion of SN-38-G to SN-38 with a half-maximal inhibitory concentration (IC50) value of 83.8 μM. PhePG and ciprofloxacin afforded the inhibition in a competitive and non-competitive manner, respectively. These findings suggest that the reduction in the serum SN-38 concentration following co-administration of ciprofloxacin during irinotecan treatment is due, at least partly, to the decreased enterohepatic circulation of SN-38 through the non-competitive inhibition of intestinal β-glucuronidase-mediated SN-38-G deconjugation.