Molecular docking and urease inhibition assay were carried out to evaluate the inhibitory activities of (-)-gossypol, gossypolone and apogossypol on Jack bean urease. The binding free energies, action sites, inhibition constants and hydrogen bonds were predicted by molecular docking study. From the docking data, the binding free energies of the three compounds were -4.39 kcal/mol, -4.91 kcal/mol and -7.07 kcal/mol, respectively. And the estimated inhibition constant of the three compounds were 607.51, 251.73 and 6.57, respectively. In addition, the inhibition assay indicated that these compounds showed varying degree of urease inhibitory activity. From the experimental results, the half maximal inhibitory concentration of (-)-gossypol, gossypolone and apogossypol were 110 μM, 51.7 μM and 9.8 μM, correspondingly. On the basis of the docking and the experimental results, both of them indicated that the hydroxyl groups of (-)-gossypol, gossypolone and apogossypol played a key role in inhibiting Jack bean urease activity, especially 6,6′-OH and 7,7′-OH.