Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) is an extracellular enzyme capable of producing cyclodextrins through an intramolecular transglycosylation reaction. With the application of cyclodextrins expanding in the industries related to food, pharmaceuticals, cosmetic, etc, CGTase has become the focus of scientific research nowadays. Calcium binding sites widely exit in α-amylase family. Previous studies indicated that these sites had very important roles for α-amylase. It was known that CGTases also possess two or three calcium binding sites. However, their structure and function are not very clear. In the present study, structure and function of calcium binding sites in CGTases were analyzed. Sequence comparisons were performed using the ClustalX 1.8 sequence alignment program. Based on the results and crystal structure analysis, it was found that calcium binding sites CaⅠ and CaⅡ exist commonly in CGTase. Most amino acids at these two calcium binding sites are highly conserved, but the residue 29 at CaⅠ and residue 199 at CaⅡ have significant differences between different types of CGTases. The residue 29 in α-CGTase primarily producing α-cyclodextrin or γ-CGTase primarily producing γ-cyclodextrin is Asp, while others are Asn. The residue 199 in γ-CGTase is Ser, while others are Asp. Calcium binding site CaⅢ only exists in few CGTases. The site consists of residues 315 and 577. In addition, site-directed mutagenesis was used to investigate the functions of calcium binding sites in CGTases. The replacement of Asp29 by Asn and Arg resulted in 23% and 35% increase in β-cyclization activity, respectively. Mutant D29R and D315A showed higher stability than wild-type CGTase at 60℃. Moreover, the mutant D315A had higher β- and γ-cyclodextrin specificity. These results suggested that calcium binding sites might be related to cycling activity, thermal stability, and product specificity of CGTases, which provided the directions for further revealing biological functions of calcium binding sites of CGTases.