Tanshinone I and its analogue dihydrotanshinone I are the major active components isolated from Salvia miltiorrhiza Bunge and Salvia Przewalskii Maxim. These compounds have been found to possess significant antibacterial, anti-dermatophytic, antioxidant, anti-inflammatory and anticancer activities. Fifteen phase I metabolites and two phase II metabolites of tanshinone I and dihydrotanshinone I in rat bile were elucidated and identified by a sensitive HPLC–ESI-MS n method. The molecular structures of the metabolites are presented on the basis of the characteristics of their precursor ions, product ions and chromatographic retention times. The results indicate that the phase I metabolites are biotransformed through four main pathways: dehydrogenation, hydroxylation, furan ring cleavage and oxidation metabolism. Phase II metabolites were mainly identified as the sulfated conjugates which showed a characteristic neutral loss of 80 Da. The biotransformed pathways of tanshinone I and dihydrotanshinone I were proposed on the basis of the investigation.