Aristolochic acid I (AAI) is widely recognized as a genotoxic and cytotoxic compound. To rationally propose detoxification strategies, it is essential to fully elucidate the in vivo disposition of AAI. Nevertheless, the toxicokinetic characteristics of AAI, particularly the possible involvement of the recirculation process, remain incompletely understood. In this research, toxicokinetics of AAI was studied following a single oral administration of AAI in Fisher rats (10, 30 and 100 mg/kg, n = 6). A method of ultra-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UPLC-QQQ-MS/MS) was developed to achieve the quantitation of AAI in rat plasma. Plasma concentration–time profiles and kinetic parameters were analyzed to characterize the toxicokinetic behavior of AAI. A secondary elevation was observed in the plasma concentration–time profiles of AAI, suggesting the existence of AAI reabsorption. The non-linear elimination kinetics of AAI might be attributed to capacity-limited excretion via bile. Additionally, the biliary excretion of AAI and several key metabolites was also explored through qualitative analysis of bile samples. For the first time, AAI-O-glucuronide was identified in bile, providing further support for enterohepatic recirculation (EHR)-mediated reabsorption of AAI. In conclusion, these findings provided solid evidence for EHR-mediated reabsorption of AAI in rats. The recirculation process might be a key mechanism responsible for the prolonged retention of AAI. In the future, detoxification strategies targeting the EHR process could be effective approaches to minimize the systemic exposure of AAI.