Alantolactone, as the principal constituent oflnula Helenium L, has been shown various pharmacologic activities, such as anti-inflammatory and deworming. In the present study, we developed a high performance liquid chromatography (HPLC) method for the determination of alantolactone in rat plasma, and pharmacokinetics of alantolactone was investigated after intravenous and oral administrations to Wistar rats. Separation was achieved on C18 column (4.6 mm×250 mm, 5.0 μm) using a mobile phase consisting of methanol-water (70:30, v/v) at a flow rate of 1.0 mL/min. The wavelength of the ultraviolet detector was set at 239 nm. The excellent linearity was found over a concentration range of 0.08-10 μg/mL (R2 = 0.9998). The intra- and inter-day precisions were good, and the RSD was lower than 2.27%. The mean absolute recovery of alantolactone in plasma ranged from 88.09% to 95.57%. After intravenous administration, alantolactone showed rapid systemic clearance (CL (0.11±0.014) L/h/kg) and small volume of distribution (Vd (0.71±0.14) L/kg). The biological half life (t1/2) was 56.24 min. After oral administration, alantolactone showed rapid oral absorption in rats, with a short Tmax of (45.02±0.88) and (45.13±0.39) min for 14 and 28 mg/kg, respectively. The bioavailability of alantolactone in rats was 50.88%, indicating that alantolactone was orally available.