The effects of difructose anhydride III (di-D-fructofuranose-1,′:2,3′-dianhydride; DFA III) administration (3% DFA III for 4 weeks) on rat intestinal microbiota were examined using denaturing gradient gel electrophoresis (DGGE). According to DGGE profiles, the number of bacteria related to Bacteroides acidofaciens and uncultured bacteria within the Clostridium lituseburense group decreased, while that of bacteria related to Bacteroides vulgatus, Bacteroides uniformis and Ruminococcus productus increased in DFA Ill-fed rat cecum. In the cecal contents of DFA III-fed rats, a lowering of pH and an increase in short chain fatty acids (SCFAs), especially acetic acid, were observed. The DFA III-assimilating bacterium, Ruminococcus sp. M-1, was isolated from the cecal contents of DFA III-fed rats. The strain had 98% similarity with R. productus ATCC 27340(T) (L76595), and mainly produced acetic acid. These results confirmed that the bacteria harmful to host health were not increased by DFA III administration. Moreover, DFA III stimulated the growth of Ruminococcus sp. M-1 producing acetic acid, which may alter the intestinal microbiota towards a healthier composition. It is expected that DFA III would be a new candidate as a prebiotic.