Objective: Although main components of the venoms from Viperidae snakes are hemotoxins, several studies indicate the presence of neurotoxins in these venoms. We previously found that the venom of pit viper Gloydius saxatilis inhibited the muscle-type nicotinic acetylcholine receptor (nAChR). The objective of present work is to isolate and characterize a neurotoxic protein from this venom. Methods: The protein was isolated by liquid chromatography and characterized using high resolution mass-spectrometry. Results and Discussion: The isolated protein called glosaxin inhibited the binding of the α-bungarotoxin to the nAChR of muscle type from Torpedo californica. Investigation of the amino acid sequence of the isolated protein by high resolution mass spectrometry and the subsequent bioinformatic analysis showed that it is homologous to the amino acid sequences of disintegrin-like proteins, consisting of non-catalytic domains of class PIII metalloproteinases from the venom of pit vipers of genus Gloydius. Glosaxin was shown to inhibit the binding of α-bungarotoxin to T. californica nAChR with IC50 = 51 μM. It also inhibited ACh-induced functional responses of the human neuronal nAChR of α3β2 subtype. Conclusions: This is the first evidence for the ability of proteins consisting of non-catalytic domains of snake venom class PIII metalloproteinase to inhibit the nAChR.