24-nor-ursodeoxycholic acid (norUDCA) is a novel therapeutic approach to cholestatic liver diseases. In mouse models of cholestasis, norUDCA induces basolateral multidrug resistance–associated proteins 4 (Mrp4) and 3 in hepatocytes, which provide alternative escape routes for bile acids accumulating during cholestasis but could also result in altered hepatic disposition of concomitantly administered substrate drugs. We used positron emission tomography imaging to study the influence of norUDCA on hepatic disposition of the model Mrp4 substrate [18F]ciprofloxacin in wild-type and Mdr2(−/−) mice, a model of cholestasis. Animals underwent [18F]ciprofloxacin positron emission tomography at baseline and after norUDCA treatment. After norUDCA treatment, liver-to-blood area under the curve ratio of [18F]ciprofloxacin was significantly decreased compared to baseline, both in wild-type (−34.0 ± 2.1%) and Mdr2(−/−) mice (−20.5 ± 6.0%). [18F]Ciprofloxacin uptake clearance from blood into liver was reduced by −17.1 ± 9.0% in wild-type and by −20.1 ± 7.3% in Mdr2(−/−) mice. Real-time PCR analysis showed significant increases in hepatic Mrp4 and multidrug resistance–associated protein 3 mRNA after norUDCA. Transport experiments in organic anion transporting polypeptide (OATP)1B1-, OATP1B3-, and OATP2B1-transfected cells revealed weak transport of [14C]ciprofloxacin by OATP1B3 and OATP2B1 and no inhibition by norUDCA. In conclusion, our data suggest that changes in hepatic [18F]ciprofloxacin disposition in mice after norUDCA treatment were caused by induction of basolateral Mrp4 in hepatocytes.