Buagafuran (BF), derived from alpha-agarofuran, is a promising anti-anxiety drug in phase I clinical trials. The present study was undertaken to examine the regulation of BF on liver cytochrome P450 (CYP) isoforms in rats. After being administered (4, 16, and 64 mg/kg) by gavage for 7 continuous days, the activities of CYP isoforms were measured by the qualification of six metabolites from CYP probe substrates using LC-MS/MS analysis. The mRNA and protein levels of CYPs were detected by reverse transcription polymerase chain reaction and Western blotting assay, respectively. Using phenacetin and chlorzoxazone as probe drugs, the activities of CYP1A2 and CYP2E1 were monitored in vivo. The result indicated that BF significantly increased the activity and protein levels of CYP1A2 and CYP2E1, while the mRNA levels were elevated to a certain extent. CYP2C6 and CYP2C11 were also slightly induced by BF, but no effect on liver CYP3A was detected in rats. Treatment of BF orally resulted in the decreasing of AUC, MRT and increasing of CL/F of phenacetin as well as production of acetaminophen in rats. The similar pharmacokinetic changes were also observed when using chlorzoxazone as a probe drug. Collectively, BF has inducing potential of liver CYP1A2 and CYP2E1 and may influence the corresponding pharmacokinetics of other drugs.