Matriptase, initially isolated from human breast cancer cells in culture, is a member of the emerging class of type II transmembrane serine proteases. Matriptase blockade could potentially modulate tumorigenesis and metastasis in vivo. Sunflower trypsin inhibitor-1 (1, SFTI-1), isolated from sunflower seeds, exhibits very potent matriptase inhibitory activity. On the basis of these findings, we designed and synthesized 13 analogues of the naturally occurring peptide 1 with the intention to explore the structure-activity relationships of this type of bicyclic peptides and to improve inhibitory selectivity and metabolic stability of the disulfide-bridge-containing peptide 1. We discovered that the methylenedithioether-bridged compound 14 demonstrates very potent binding affinity to matriptase. Compound 8 exhibits much better selectivity for inhibition of matriptase versus thrombin, whereas compound 2 becomes a more potent thrombin inhibitor, which can be potentially used as an anticoagulant for prophylaxis and therapy of thromboembolism.