Silibinin is a mixture of two flavonoid lignan silibinins A and B from the seeds of milk thistle (Silybum marianum L.). Using ultra‐performance liquid chromatography/quadrupole time‐of‐flight–MS (UPLC/Q‐TOF–MS), a total of 18 metabolites were identified in rat and human urine samples after oral administration of silibinin capsule. Furthermore, nine glucuronides and/or sulfated metabolites and two prototype compounds were simultaneously quantified in rat urine after oral administration of silibinin capsule at 50 and 100 mg/kg. Over a 72‐h period, 27.6% and 23.3% of silibinin were excreted in the form of metabolites (n = 11) in urine, among which five major metabolites, including silibinin A‐7‐O‐β‐glucuronide (SA‐7G), silibinin B‐7‐O‐β‐glucuronide (SB‐7G), silibinin A‐5‐O‐β‐glucuronide (SA‐5G), silibinin B‐5‐O‐β‐glucuronide (SB‐5G) and silibinin A‐20‐O‐glucuronide (SA‐20G), accounted for 20.5% and 15.5% of the dosages, respectively, when administered at doses of 50 and 100 mg/kg. These results suggested that glucuronidation at the C7‐, C5‐ and C20‐hydroxyls was the primary metabolic pathway of silibinin diastereoisomers in vivo. The present results provide helpful information about the in vivo metabolism and clinical usage of silibinin capsule.