We investigated the influences of difructose anhydride III (DFAIII), a novel commercially available disaccharide, on sugar metabolism, breath hydrogen and serum acetate in the early stages after ingestion to determine whether DFAIII is an indigestible sugar and to what degree it is fermentable in humans. This study was designed as a randomized controlled single-blind crossover test with 9 healthy subjects, who drink a 200 mL water solution containing 10 g of DFAIII, lactulose or sucrose following overnight fasting. Blood samples (for analysis of glucose, fructose, insulin, triacylglycerol, free fatty acids, and acetate) were collected at 0, 0.5, 1, 2, 4, 8 h after the ingestion and breath samples (for analysis of hydrogen and methane gases) were collected at 1 h intervals until 8 h after the ingestion. We also interviewed each subject hourly about the incidence and severity of specific abdominal complaints and other symptoms. The results revealed that ingestion of 10 g of DFAIII did not change the serum levels of glucose, fructose, and insulin, similarly to the case with lactulose, and no increase in breath hydrogen excretion was comparable to the case with sucrose. The incidence of specific abdominal symptoms tended to be lower after DFAIII ingestion than after lactulose ingestion. It thus turned out that DFAIII was indigestible and low fermentable in the early stages after ingestion.