In the study, we investigated the contribution of Ca²⁺ to the thermostability of α-cyclodextrin glycosyltransferase (α-CGTase) from Paenibacillus macerans , which has two calcium-binding sites (CaI and CaII), and β-CGTase from Bacillus circulans , which contains an additional calcium-binding site (CaIII), consisting of Ala315 and Asp577. It was found that the contribution of Ca²⁺ to the thermostability of two CGTases displayed a marked difference. Ca²⁺ affected β-CGTase thermostability significantly. After Ca²⁺ was added to β-CGTase solution to a final concentration of 5 mM followed by incubation for 120 min at 60 °C, residual activity of β-CGTase was 88.3%, which was much higher than that without Ca²⁺. However, Ca²⁺ had a small contribution to α-CGTase thermostability. Furthermore, A315D and D577K mutations at CaIII could significantly change the contribution of Ca²⁺ to β-CGTase thermostability. These results suggested that the contribution of Ca²⁺ to CGTase thermostability was closely related to CaIII.