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Thrombin, which plays a crucial role in hemostasis, is also implicated in cancer progression. We investigated the effects of the thrombin-targeting recombinant tyrosine-sulfated madanin-1 on cancer cell behavior and signaling pathways compared with wild-type (WT) madanin-1. We generated recombinant madanin-1 2 sulfation (2S) and madanin-1 WT proteins using E. coli. SKOV3 and MDA-MB-231 cells were treated with purified recombinant proteins with or without thrombin stimulation. Migration and invasion of cells were analyzed by a wound healing assay and transwell assay, respectively. Thrombin markedly increased cell migration and invasion in both SKOV3 and MDA-MB-231 cells, which were significantly suppressed by madanin-1 2S (p p p in vivo validation is required.