We studied the mutation effect of subsites -3(Lys47), -7(146-152), and cyclization center (Tyr195) in active domain on product specificity of α-cyclodextrin glucanotransferase (α-CGTase) from Paenibacillus macerans sp. 602-1. The Lys47 was replaced by Thr47 and Tyr195 by Ile195, and the amino acids from 146 to 152 were replaced by Ile (named as Δ6). All these mutant α-CGTases were actively expressed in E. coli BL21. Compared with the wild-type α-CGTase, the starch-degrading activities of all the mutant enzymes were declined. For mutant Y195I, the percentage of α-CD was decreased from 68% to 30%, and β-CD was raised from 22.2% to 33.3%. Interestingly, γ-CD was increased from 8.9% to 36.7% and became the main product, while the actual yield was increased from 0.4 g/L to 1.1 g/L. Mutant K47T and Δ6 still produced α-CD as main product though the percentage of β- and γ-CD increased. Purified Y195I CGTase showed similar optimum temperature with the wild-type α-CGTase, but its optimum pH shifted from 5.0 to 6.0 with better pH stability. In summary, mutant Y195I CGTase has the potential to produce γ-CD as the main product.