The cDNA of gloshedobin was synthesized and amplified by RT-PCR from the total RNA of snake (Gloydius shedaoensis) venom gland. The 711 bp nucleotide sequence, which encodes the mature gloshedobin, was cloned into expression vector pPIC 9K and transferred into yeast Pichia pastoris, strain GS115. Transfermants with phenotype His⁺Mut⁺ were selected to study their expression. Recombinant protein was conveniently separated and purified from the supernatant by two chromatographic steps: ion exchange chromatography on Q-Sepharose FF and affinity chromatography on Benzamidine-Sepharose 4BCL. Like intact gloshedobin, the recombinant enzyme exhibited strong esterase activity using tripeptide p-nitroanilide derivatives as substrate, but hydrolyzed N-p-tosyl-L-arginine methyl ester (TAME) very weakly. The recombinant protein displayed extreme instability at 37℃ in neutral buffer but higher stability at 0℃, and also, pH is not a key factor to affect its stability while the optimal pH for its enzymatic activity is pH 8.0.