Hemorrhagic toxin (TcsH) is an important exotoxin produced by Paeniclostridium sordellii, but the exact role of TcsH in the pathogenesis remains unclear, partly due to the lack of knowledge of host receptor(s). Here, we carried out two genome-wide CRISPR/Cas9 screens parallelly with TcsH and identified cell surface fucosylation and TMPRSS2 as host factors contributing to the binding and entry of TcsH. Genetic deletion of either fucosylation biosynthesis enzymes or TMPRSS2 in the cells confers resistance to TcsH intoxication. Interestingly, TMPRSS2 and fucosylated glycans can mediate the binding/entry of TcsH independently, thus serving as redundant receptors. Both TMPRSS2 and fucosylation recognize TcsH through its CROPs domain. By using Tmprss2?/? mice, we show that Tmprss2 is important for TcsH-induced systematic toxicity and colonic epithelial lesions. These findings reveal the importance of TMPRSS2 and surface fucosylation in TcsH actions and further provide insights into host recognition mechanisms for large clostridial toxins.