The digestive protease cathepsin B1 (SmCB1) of the human blood fluke Schistosoma mansoni is a potential drug target for the treatment of schistosomiasis, a parasitic disease that afflicts over 200 million people worldwide. SmCB1 is biosynthesized as an inactive zymogen in which the pro-peptide operates as an intra-molecular inhibitor by blocking the active site. We have investigated the activation processing through which the pro-peptide is proteolytically removed and the regulatory role of sulfated polysaccharides (SPs) in this process. Crystal structures of three molecular forms of SmCB1 along the activation pathway were determined, namely the zymogen, an activation intermediate with partially cleaved pro-peptide, and the mature enzyme. We have demonstrated that SPs are essential for the autocatalytic activation of SmCB1 as they interact with a specific heparin-binding domain in the pro-peptide inducing its complete processing. An alternative activation route for SmCB1 is mediated by S. mansoni legumain and is down-regulated by SPs, indicating that SPs act as a molecular switch between both activation mechanisms.