Inulin is a widespread polysaccharide, and it can be bio-transferred to α-d-fructofuranose-β-d-fructofuranose 1,2':2,1'-dianhydride (DFA I), a potential low-calorie sweetener, via inulin fructotransferase (IFTase, EC 22.214.171.124). In this study, a novel DFA I-forming IFTase was identified and characterized from Streptomyces peucetius subsp. caesius ATCC 27952 (SpIFTase). The specific activity of SpIFTase was determined to be 420.84 ± 6.21 U mg-1 at pH 6.5 and 45 °C. The enzyme exhibited a prominent thermostability with a half-life of 70 min at 70 °C and a structural melting temperature (Tm) of 75.48 °C. The values of Km and kcat/Km of SpIFTase against inulin substrate were 3.08 mM and 199.09 mM-1 s-1, respectively. Furthermore, the catalytic residues were investigated by site-directed mutagenesis with an alanine scanning method. Interestingly, the critical catalytic residues of SpIFTase were speculated to be residues D162 and E231, which were firstly non-conserved with those of previously reported IFTases. This work proposes a potential for industrial DFA I production and new insights into the catalytic mechanism of DFA I-forming IFTases.