To make a clear understanding of the in vivo metabolism of propyl gallate in rats and determine the original compounds of the metabolites of Paeoniae Radix Rubra (PRR), the urine and plasma of the rats administrated with propyl gallate were collected, and then the HPLC-DAD-ESI-IT-TOF-MS(n) technique was applied to screen and identify the metabolites of propyl gallate from these bio-samples. By comparing the collected LC-MS data, the origin of the metabolites of PRR were confirmed. Finally, 33 metabolites of propyl gallate were identified from urine sample, and 20 metabolites of propyl gallate were identified from the plasma sample. In total, 37 metabolites of propyl gallate were identified, and 17 of them were identical to the metabolites of PRR. They are mainly the phase II metabolites of propyl gallate, 3-hydroxypropyl 3,4,5-trihydroxybenzoate, gallic acid and pyrogallol. Phase I reactions (decarboxylation, hydroxylation) and phase II reactions (sulfation, glucuronidation and methylation) were observed as the main metabolic pathways of propyl gallate. In this research, the in vivo metabolism of proply gallate was reported for the first time and 37 metabolites were identified, among which 35 were new metabolites of propyl gallate, and 20 were new compounds. The results demonstrated that 17 metabolites of PRR can be formed from propyl gallate. This will enhance our understanding of the metabolism and Effective forms (the truly active structures) of propyl gallate and PRR.