Dihydromyricetin (DMY), a flavanonol compound found as the most abundant and bioactive constituent in vine tea (Ampelopsis grossedentata), possesses numerous biological activities. In the present study, a HPLC-MS/MS method for the determination of DMY in tissues, urine and feces was developed and applied to the tissue distribution and excretion study after oral administration in rats, and the metabolic profile of DMY was further investigated using UPLC-QTOF-MS. The results indicated that DMY could be distributed rapidly in various tissues and highly in gastrointestinal tract. The elimination of DMY occurred rapidly as well, and most unconverted forms were excreted in feces. A total of eight metabolites were identified in urine and feces, while metabolites were hardly found in plasma. The predicted metabolic pathways including reduction, dehydroxylation, methylation, glucuronidation and sulfation were proposed. The present findings may provide the theoretical basis for evaluating the biological activities of DMY, and will be helpful for its future development and application.