Abstract 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 < 0.05). Madanin-1 2S also significantly suppressed thrombin-induced expression of phosphorylated Akt and extracellular signal-regulated kinase (ERK) in both cell lines (p < 0.05), but not by madanin-1 WT in MDA-MB-231 cells. Furthermore, madanin-1 2S significantly reversed the expression of E/N-cadherin and vimentin in thrombin-treated MDA-MB-231 cells (p < 0.05), whereas madanin-1 WT did not show any effect. In conclusion, madanin-1 2S suppressed migration and invasion of cancer cells more effectively than madanin-1 WT. We postulate that inhibiting thrombin via the sulfated form of madanin-1 may be a potential candidate for enhanced cancer therapy, albeit further in vivo validation is required.