Objective Explore euscaphic acid uptake and transport mechanisms in Caco-2 cells. Methods Ultra-high performance liquid chromatography-triple quadrupole-mass spectrometry ( UPLC-TQ-MS) was employed to determine the content of euscaphic acid. The effects of various times and temperatures on its uptake were investigated. On the basis of the result of uptake analyses, the effects of various concentrations, P-gp inhibitors, chelating agents, and pH values on its bidirectional transport were explored. Results Euscaphic acid uptake was (8. 38±0. 87) μg/ mg for 180 min in Caco-2 cells at 37℃. The apparent permeability coefficient (Papp ) values of euscaphic acid at low, medium, and high concentrations were (61. 41±2. 92) ×10-4, (146. 90±14. 91) ×10-4, and (167. 18±6. 72) ×10-4 cm/ s, respectively, which were positively correlated to the concentrations. P-gp inhibitors and chelating agents had no effect on Papp values. A weakly acidic environment (pH= 6) significantly increased the Papp value, and the efflux rate ranged from 0. 8 to 1. 4. Conclusions Euscaphic acid has good transmembrane permeability in Caco-2 cells, and the uptake mode is mainly passive diffusion. It is not a substrate of P-gp and no cellular bypass transport exists. This study may provide an experimental basis for in vivo intestinal absorption of medicines containing euscaphic acid.