Gentiopicrin is the main component of the famous Chinese patent medicine Long Dan Xie Gan Wan, and belongs to iridoid glycoside with a variety of pharmacological activities, such as liver protection, liver damage prevention. However, it has been reported that gentiopicrin is quickly absorbed in vivo and has low bioavailability in mice. As we known, the types of metabolic reactions of intestinal bacteria are different from that of liver metabolism, and intestinal bacteria play an important roles for the efficacy of compounds with low oral bioavailability. Thus, it is necessary to better understand the metabolic pathway of gentiopicrin. In the work, we adopted the co-incubation system of intestinal bacteria and gentiopicrin. Six metabolites were analyzed and identified, which includes aglycone reduction product (G-M1 and G-M2), aglycone hydrolysis product (G-M3), erythrocentaurin reduction product (G-M4 and G-M6), and dehydration product (G-M5). Three metabolites (G-M3, G-M5 and G-M6) have not been previously reported. We also demonstrated for the first time that G-M1 and G-M2 were chiral metabolites of gentiopicrin by NMR data. Moreover, possible new metabolic pathways of gentiopicrin have been found. Therefore, this study provides a new clue to the type of metabolic reactions and the spectrum of metabolites of gentiopicrin.