Glycosides are important active components in traditional Chinese medicine (TCM). Their pharmacological activity, pharmacokinetic characteristics and in vivo existence become hotspots of current studies. The metabolic pathways of these glycosides are de-glycosylation mainly mediated by gut microbiota. After glycosides were metabolized into aglycones, they could be absorbed more easily and show better pharmacological effects. In this article, we reviewed the main glycosidase in gut microbiota which helps metabolize TCM glycosides, relevant bacterial strains which generate glycosidase, as well as the de-glycosylated metabolic pathways of the representative glycosides, on the basis of gut microbiota's important roles in in vivo metabolism and efficacy of TCM glycosides. We also preliminarily solved problems in studies on de-glycosylation of TCM glycosides.