A reversed-phase high performance liquid chromatographic method was established for determination of intestinal trefoil factor (ITF) in burned rats. The analysis was carried out on a Hypersil C4 column (30 nm, 5 microns, 4.6 mm i.d. x 250 mm) with gradient elution of acetonitrile-trifluoroacetic acid-water solution at a flow rate of 1 mL/min and detected by a UV detector at 214 nm. The volume proportion of trifluoroacetic acid in water is 0.1% and the volume proportion of acetonitrile in mobile phase ranged from 19.5% to 42.0%. The linear range of the method was 1 mg/L-100 mg/L with relative coefficient of 0.9989. The minimum detection limit was 0.5 mg/L. The recovery of ITF added ranged between 93.95% and 105.90%. The intra-day and inter-day RSD of ITF were less than 5.33% and 6.10% respectively. This method is precise, accurate and can be used for the determination of ITF in intestinal mucosal of burned rats. Therefore, it is helpful in the research of comparing the effects of enteral feeding and parenteral nutrition on ITF expression.