A simple HPLC–UV method was established for the determination of orientin in plasma and different tissues of rat (heart, liver, spleen, lung, kidney, brain, stomach and small intestine). The separation was achieved by HPLC on a C18 column with a mobile phase composed of acetonitrile–0.1% acetic acid (20:80, v/v), UV detection was used at 348 nm. Good linearity was found between 0.250–50.0 μg/ml (r2 = 0.9966) for plasma samples and 0.050–50.0 μg/ml (r2 ≥ 0.9937) for the tissue samples, respectively. Within- and between-day precisions expressed as the relative standard deviation (R.S.D.) for the method were 2.3–9.6% and 3.0–7.4%, respectively. The relative recoveries of orientin ranged from 95.4 to 100.6% for plasma and 93.1 to 107.9% for tissue homogenates. The developed method was successfully applied to the pharmacokinetics and tissue distribution research after intravenous administration of a 20 mg/kg dose of orientin to healthy Sprague–Dawley rats. The main pharmacokinetics parameters obtained presented that orientin was quickly distributed and eliminated within 90 min after intravenous administration. The tissue distribution results showed that liver, lung and kidney were the major distribution tissues of orientin in rats, and that orientin had difficulty in crossing the blood–brain barrier. It was also found that there was no long-term accumulation of orientin in rat tissues.