Abstract: The aim of this paper was to investigate the absorption mechanism of silybin(SLB)in Caco-2 cells. Concentrations of samples in the study were determined by developing LC/MS/MS method of SLB, propranolol and atenolol in HBSS buffer to calculate apparent permeability coefficient(P_app). When Caco-2 cells were cultured to the 21st day, the TEER were above 350 Ω ·cm². The P_app of Lucifer yellow was far less than 1 × 10^-7 cm/s. As the positive control drugs, The P_app of atenolol and propranolol were similar to those reported in the literature, indicating that the Caco-2 monolayer model was successfully established in this experiment. The P_app(AP-BL) of SLB at 5, 20, and 50 μg/mL were all more than 2×10^-6 cm/s, which showed that SLB was a moderately permeable drug. The efflux ratio was greater than 2 indicating the efflux transporter may be involved in the absorption process of SLB. The P_app of silybin-N-meglumine was similar to that of SLB, suggesting that salt formation did not alter the membrane permeability of SLB. In conclusion, the membrane permeability of SLB is good, and its solubility is low. SLB is a BCS class 2 drug. The release of SLB in the gastrointestinal tract is an important factor in its absorption process.