Urea transporter B (UT-B) is a membrane channel protein that specifically transports urea.UT-B null mouse exhibited urea selective urine concentrating ability deficient, which suggests the potential clinical applications of the UT-B inhibitors as novel diuretics.Identification of inhibitor binding sites in UT-B in silico lays a foundation for diuretics discovery.Primary High Throughput Virtual Screening of 50000 small-molecular drug-like compounds hit 2319 compounds.Then 2319 compounds were screened by High-Throughput Screening using an erythrocyte osmotic lysis assay.Based on the physiological data, putative UT-B binding site was identified by structure-based drug design and validated by ligand-based and QSAR model.Additionally, UT-B structural and functional differences under inhibitors treated and untreated conditions were simulated by Molecular Dynamics.As the result, we identified four classes of compounds with UT-B inhibitory activity and predicted human UT-B model, based on which computative binding site was identified and validated.A novel mechanism of differences in UT-B inhibitory activity was discovered in cross-species.Results suggest residue PHE198 in rat and mouse UT-B might block the inhibitor migration pathway.Inhibitory mechanisms of UT-B inhibitor and the functions of key residue were proposed.Binding site analysis provides structure bases for lead identification and optimization of UT-B inhibitors.