Abstract: AIM: To study the metabolites of roxithromycin (RXM) in the bile of a patient, who had received an oral dose of 150 mg RXM after gallbladder operation. METHODS: High-performance liquid chromatography/ion trap mass spectrometry was used to determinate RXM and its metabolites in human bile. The bile sample was collected, extracted with diethyl ether and separated on a C₁₈ reversed-phase column with (+)ESI-MSⁿ detection. RESULTS: RXM and its thirteen metabolites were identified in the sample based on reference substances and mass spectra. These include descladinose-RXM (M1), erythromycin-oxime (M2) and its N-mono- and N-didemethylated derivatives (M4 and M6), N-mono- and didemethylated derivatives of RXM (M3 and M5), as well as the (9Z)-epimeric isomers of RXM and all its above metabolites (M7～M13). The ratios of (9Z)- to (9E)-isomers were found to be between 0.11～0.79. The parent drug remained was about 20% of the total bile metabolites. CONCLUSION: The metabolic pathways of RXM observed in the human bile are therefore quite different to those in human plasma and urine. The identification of the isomerization of the oximes of RXM derivatives represents a novel biotransformation pathway.