[Objectives]  The aim of this study was to clone and express the venom antithrombin Ⅲ gene of Scleroderma guani (SgAT-Ⅲ) and thereby provide a basis for characterizing the physiological function of this gene. [Methods]   The open reading frame (ORF) sequence of the SgAT-Ⅲ gene was cloned by reverse transcription PCR technology and its sequence structure analyzed with bioinformatic software. The gene expression profiles of SgAT-Ⅲ in different developmental stages and tissues of female adults were detected with qPCR. The gene was expressed using the prokaryotic vector pCzn1. [Results]  The ORF of the SgAT-Ⅲ gene was successfully cloned and found to be 1 338 bp in length, encoding 446 amino acids with a signal peptide comprised of amino acids 1-20, a predicted molecular weight of 49.49 ku and an isoelectric point (pI) of 6.23. The results of multiple sequence alignment revealed that SgAT-Ⅲ shares high amino acid identity (>64%) with SgAT-Ⅲs of other members of the Formicidae. Its C-terminal sequence has a typical, reactive, center loop region of the serpin protein family, and contains an active cleavage site that can be recognized by target protease. Phylogenetic analysis indicates that SgAT-Ⅲ is closely related to AT-Ⅲs of other hymenopteran insects, particularly those of ants. qPCR analysis revealed that the SgAT-Ⅲ gene was abundantly expressed in the venom apparatus. SDS-PAGE electrophoresis detection revealed that the recombinant SgAT-Ⅲ was successfully expressed, and that high-purity recombinant SgAT-Ⅲ was obtained. [Conclusion]  The SgAT-Ⅲ gene was successfully cloned and found to be abundantly expressed in the venom apparatus. Obtaining this purified, recombinant protein lays a foundation for further investigation of the physiological function of SgAT-Ⅲ.