To determine the absorption properties and study the intestinal absorption kinetics of poncidin in rats. In situ single pass perfusion model was combined with High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS/MS) method to investigate the intestinal absorption characteristics and calculate absorption parameters with aspects of drug absorption, concentration and perfusion medium. The absorption of poncidin under acid condition at pH 6.5 was more stable, where intestinal enzymes showed little influence on metabolism, and the absorption was significantly higher than that in pH alkaline condition at pH 8.0 (P<0.05). Drug concentrations had no significant influence on absorption rate constant of the same intestinal segment Ka and apparent permeability coefficient Papp values of poncidin. Different concentrations of poncidin showed no significant differences in the Ka and Papp values among duodenum, jejunum and colon, but the values were significantly lower than ileum absorption parameters, with significant differences (P<0.05). There was no significant effect of verapamil on intestinal absorption of poncidin. The results showed that poncidin could be absorbed at all the studied intestinal segments while ileum seemed to be the best absorption segment in the concentration range of 10-1 000 μg·L⁻¹. The absorption was characterized by a linear dynamic process of passive transport, without absorption saturation. Weak acid environment was helpful for the intestinal absorption of poncidin, and ponicidin was not a substrate of P-glycoprotein (P-gp).