The purpose of this study was to investigate the effect of bile salts on gastrointestinal absorption and the plasma second peak phenomenon of roxithromycin in rats. The pharmacokinetic parameters of roxithromycin were calculated after single oral administration at a dose of 20 mg/kg in sham-operated (control), bile duct cannulated (BDC) and bile salt co-administered bile duct cannulated (BSBDC) rats. In BDC rats, the total area under the plasma concentration–time curve from time zero to time infinity (AUC0–∞) and the peak plasma concentration (Cmax) were significantly smaller (0.572-fold) and lower (0.412-fold), respectively, than those in the control rats. These values were recovered by co-administration of bile salt (0.831- and 0.828-fold for AUC0–∞ and Cmax compared with the control, respectively). Thus, the decreased absorption of roxithromycin in BDC rats could be due to a depletion of bile. The solubility of roxithromycin was 3.09-fold increased at 30 mm of sodium taurocholate. The oral dosage regimen of roxithromycin could be changed in patients with bile deficiency or when the drug is administered to individuals on a high-fat diet, if the present rat data can be extrapolated to humans. Copyright © 2013 John Wiley & Sons, Ltd.