To investigate the mechanism of high product specificity of γ-clodextrin glucanotransferase (CGTase) from alkalophilic Bacillus clarkii 7364, we aligned protein sequence and structure model, found out that loss of 6 amino acids at subsite ?7 probably affected its product specificity. Using overlapping PCR method, we inserted 6 amino acids into subsite ?7 of CGTase. The mutant CGTase gene was ligated with pET-20b (+) and expressed in Escherichia coli BL21 (DE3). The extracellular recombinant enzyme was used to transform soluble starch into cyclodextrins (CDs). HPLC analysis results show that, compared to wild CGTase, the γ-CDs produced by mutant enzyme decreased from 76.0% to 12.5%, whereas the ratio of α- and β-CDs increased from 8.7% and 15.2% to 37.5% and 50%. The possible mechanism was that, compared to α-, β-CGTase, wild γ-CGTase lacks 6 amino acids in its subsite ?7. This conformation provided more space for glucose combination and was thus advantageous for forming γ-CD. When the 6 amino acids were inserted into the subsite ?7 of wild γ-CGTase, the space to bind with glucose reduced and consequently resulted in less γ-CD production.