The kinetic characteristics of β-cyclodextrin production by a cyclodextrin glycosyltransferase (CGTase) produced by Bacillus sp. C26, a new isolate from a soil sample was investigated. Considering highest yield and initial production rate of β-cyclodextrin, among the starches examined, soluble starch, tapioca starch, sago starch, corn starch and rice starch, tapioca starch was the best substrate for this CGTase. The optimum temperature for tapioca starch gelatinization prior to its use as a substrate for β-cyclodextrin production was 65ºC. The yield and initial production rate of β-cyclodextrin increased with increasing starch concentration up to 6% and an enzyme concentration up to 48 U/g-starch. The kinetic parameters of Vmax and Km of β-cyclodextrin production from tapioca starch by CGTase were 1.59 mg/mL/h and 22.3 mg/mL, respectively. Considering high initial production rate and high yield of β-cyclodextrin, the optimum reaction temperature was at 50ºC. This study provided the necessary kinetic information that may be useful to define the most suitable condition for industrialized production ofβ-cyclodextrin with the high yield and productivity.