Objective: The root of Cynanchum auriculatum Royle ex Wight(Asclepiadaceae), a well-known traditional Chinese medicine named ‘‘baishouwu'', was reported to exhibit various biological activities. Caudatin-2, 6-dideoxy-3-O-methyl-β-d-cymaropyranoside(CDMC), a C-21 steroidal glycoside isolated from baishouwu, has been proved to possess antitumor activities in our previous studies. However, the routes and metabolites of CDMC in vivo remained largely unknown. In this study, the zebrafish model organism was used to clarify the metabolism of CDMC in vivo. Methods: The ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry(UPLC/Q-TOF-MS) technique combined with MetabolynxTM software was applied to identify the metabolites of CDMC in zebrafish after exposure for 24 h. Results: Ten metabolites of CDMC were found or tentatively identified based on MS and MS/MS data. The results suggested that hydroxylation, hydrogenation, dehydrogenation, demethylation, dehydroxylation and deglycosylation were the main metabolic processes of CDMC in zebrafish. Conclusion: Our study certified that the zebrafish model can wonderfully imitate the current models in explaining metabolic pathways of steroid compounds with the advantages of lower cost, far less amount of compounds needed, easy operation and high performance.