Matriptase, a trypsin-like serine protease, which may be involved in tissue remodeling, cancer invasion, and metastasis. Potent and selective matriptase inhibitors not only would be useful pharmacological tools for further elucidation of the role of matriptase in these processes but also could have therapeutic potential for the treatment and/or prevention of cancers. We report herein the structure-based approach for the discovery of bis-benzamidines as a novel class of potent matriptase inhibitors. The lead compound, hexamidine (1), inhibits not only the proteolytic activity of matriptase, (K(i) = 924 nM) but also of thrombin K(i) = 224 nM). By testing several available analogues, we identified a new analogue (7) that has a K(i) = 208 nM against matriptase and has only weak inhibitory activity against thrombin (K(i) = 2670 nM), thus displaying a 13-fold selectivity toward matriptase. Our results demonstrated that structure-based database screening is effective in the discovery of matriptase inhibitors and that bis-benzamidines represent a class of promising matriptase inhibitors that can be used for further drug design studies. Finally, our study suggested that there is sufficient structural differences between matriptase and its closely related serine proteases, such as thrombin, for the design of potent and selective matriptase inhibitors.