OBJECTIVE： To establish a method for the concentration determination of nateglinide in rats’ plasma and study its pharmacokinetic characteristics in rats in vivo. METHODS： UPLC was performed on the column of Acquity UPLC® BEH C18 with mobile phase of acetonitrile-10 mmol/L potassium dihydrogen phosphate buffer （41 ∶ 59， V/V） at flow rate of 0.38 mL/min， with column temperature of 35 ℃， detection wavelength of 210 nm and volume of 2 μL. 18 Wister rats were intragastrically administrated nateglinide 16 mg/kg. Blood sample 0.4 mL was taken from medial canthus before administration and after 10， 20， 30， 45， 60， 90， 120， 180， 240， 360， 480 min of administration. The concentration of nateglinide in rats’ plasma was determined； then DAS 2.1.1 software was used to calculate its pharmacokinetic parameters. RESULTS： Nateglinide showed good linear relationship in 0.05-6.4 μg/mL （r＝0.999 3）， lower limit of quantification was 0.05 μg/mL； RSDs of inter-day （n＝5） ， intra-day （n＝3） and stability （n＝3）  tests were lower than 10％； extraction recovery rate and method recovery rate were 78.71％-80.56％， 91.78％- 100.42％ （RSD＜10％， n＝5）， respectively. After rats were intragastrically administrated nateglinide， AUC0-8 h was （5.87±2.32）    μg·h/mL， AUC0-∞ was （6.11±2.48） μg·h/mL， t1/2 was （1.72±0.55） h， tmax was （0.67±0.29） h and cmax was （3.34±1.23） μg/mL. CONCLUSIONS： The method is accurate， rapid with strong specificity， and can be used for the concentration determination of nateglinide in rats in vivo； nateglinide is absorbed and metabolized quickly in rats in vivo.