As a popular beverage, tea (Camellia sinensis, Theaceae) has been associated with many beneficial health effects, including the prevention of cancer and heart disease, a phenomenon mostly attributed to the presence of polyphenolic compounds. The main bioactive polyphenols in black tea are theaflavins, which are formed by the oxidation and polymerization of catechins in green tea leaves during fermentation. Theaflavins including theaflavin (TF), theaflavin‐3‐gallate (TF3G), theaflavin‐3′‐gallate (TF3′G), and theaflavin‐3,3′‐digallate (TFDG). We and others have found that theaflavins have poor systematic bioavailability. Therefore, it is still unclear how these compounds could exert their biological functions. It has been reported that higher molecular weight polyphenols are metabolized by the microbiota and their metabolites may play an important role in chronic disease prevention. In the present study, we investigated the microbial bioconversion of theaflavins in specific pathogen free (SPF) mice, germ free (GF) mice, and in human fecal microbiota in vitro. Microbial bioconversion was monitored using liquid chromatography/electrospray ionization tandem mass spectrometry by analyzing the MSn (n = 1–3) spectra. Our results indicated that TFDG, TF3G and TF3′G can be metabolized by gut microbiota to generate gallic acid, pyrogallol, and TF. Glucuronidated and sulfated, instead of methylated metabolites of TF3G, TF3′G and TFDG were detected and identified as the minor mouse fecal metabolites of TFDG. To our knowledge, this is the first report on the microbial metabolism of theaflavins.