We produced high purity difructose anhydride III (DFA III; α-D-fructofuranose-β-D-fructofuranose 1,2´: 3´,2 dianhydride) crystals from the crude inulin extracted from chicory with inulin fructotransferase (depolymerizing) induced by H65-7 strain of Arthrobacter sp. The physical, chemical, and physiological properties of DFA III have been investigated in this report. The degradability and Maillard reactivity of DFA III in the low pH solutions were much lower than those of sucrose. DFA III showed no utilization by Streptococcus mutans and Bifidobacterium in vitro. Digestion test with rat intestinal acetone powder and absorption test with everted sacs of rat small intestine showed that DFA III had low digestibility and absorbability in vitro. Diet containing 2% (w/w) DFA III increased the cecal short chain fatty acids and induced a shift of the cecal microflora in rats. On the other hand, a daily oral ingestion of 5 g DFA III by healthy adults showed no influence on fecal microflora.