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An antihemorrhagic protein has been isolated from the serum of Chinese mamushi (Gloydius blomhoffi brevicaudus) by using a combination of ethanol precipitation and a reverse-phase high-performance liquid chromatography (HPLC) on a C8 column. This protein-designated Chinese mamushi serum factor (cMSF)-suppressed mamushi venom-induced hemorrhage in a dose-dependent manner. It had no effect on trypsin, chymotrypsin, thermolysin, and papain but inhibited the proteinase activities of several snake venom metalloproteinases (SVMPs) including hemorrhagic enzymes isolated from the venoms of mamushi and habu (Trimeresurus flavoviridis). A similar protein (Japanese MSF, jMSF) with antihemorrhagic activity has also been purified from the sera of Japanese mamushi (G. blomhoffi). The N-terminal 70 and 51 residues of the intact cMSF and jMSF were directly analyzed; a similarity between the sequences of two MSFs to that of antihemorrhagic protein (HSF) from habu serum was noticed. To obtain the complete amino acid sequences of MSFs, cDNAs encoding these proteins were cloned from the liver mRNA of Chinese and Japanese vipers based on their N-terminal amino acid sequences. The mature forms of both MSFs consisted of 305 amino acids with a 19-residue signal sequence, and a unique 17-residue deletion was detected in their His-rich domains.