Highlights ► We develop an HPLC/MS method for quantification of atractylenolide I in rat plasma. ► Pharmacokinetics was performed in rats ingested with 20 g/kg Atractylodis extract. ► Atractylenolide I was found to be absorbed quickly within 1 h. ► The biological half-life and the elimination rate constant of atractylenolide I were 1.94 ± 0.27 h and 0.365 ± 0.06 h, respectively.