Abstract: In vitro anaerobic culture of rat gut microbiota with berberine was investigated  and the UPLC-Q-TOFMS/MS was used to analyze berberine metabolites and its biotransformation process. Berberine and its metabolites could be isolated effectively with UPLC, then tandem mass spectrometer was used to analyze and identify metabolites’ structures.The results showed that rat gut microbiota could transform berberine into 5 metabolites such as berberrubine and jatrorrhizine through demethylation, dehydroxylation, demethylene reactions in vitro.