The possible inclusion modes of berberine(Berb) with β-cyclodextrin(BCD) in aqueous solution were predicted by molecular docking,molecular dynamics(MD) simulation and binding free energy calculations.Firstly,the molecular docking result reveals that the docking conformations of Berb appeared in two clusters ranked in two opposite orientations.Then,10 ns MD simulation was performed separately on the lowest energy conformation of each orientation(Mode I and Mode II) obtained by molecular docking.Moreover,based on the trajectories from MD simulation,the binding free energies of the two different modes were calculated by the Molecular Mechanics/Poisson Boltzmann surface area(MM/PBSA) method.Through analyzing the binding free energies of Berb with BCD,we found that Mode II was the preferential inclusion mode,which was in good agreement with the experimental result.In addition,the computed results show that the main impetus for the complex was the van der Waals interaction,but the solvation energy and the entropy change produced an adverse effect on the complex. The possible inclusion modes of berberine (Berb) with β-cyclodextrin (BCD) in aqueous solution were predicted by molecular docking, molecular dynamics (MD) simulation and binding free energy calculations. Firstly, the molecular docking result reveals that the docking conformations of Berb found in two clusters ranked in two opposite orientations. Ten, 10 ns MD simulation was performed separately on the lowest energy conformation of each orientation (Mode I and Mode II) obtained by molecular docking. Moreover, based on the trajectories from MD simulation, the binding free energies of the two different modes were calculated by the Molecular Mechanics / Poisson Boltzmann surface area (MM / PBSA) method. Through analyzing the binding free energies of Berb with BCD, we found that Mode II was the preferential inclusion mode, which was in good agreement with the experimental result. In addition, the computed results show that the main impetus for the complex was the van der Waals interaction, but the solvation energy and the entropy change produced an adverse effect on the complex.