Lesser yam (Dioscorea esculenta L.) is one of the many types of Dioscorea spp. growing in Indonesia and its tuber contain the highest amount of inulin (14.77% db) among others (Winarti et al., 2011). The tuber is very important as an alternative source of carbohydrate in the countryside. Isolation and characterization of inulin from lesser yam tubers have been performed. The aim of study was investigated the effect of lesser yam inulin to change of the colonic micro biota (Bifidobacteria, Lactobacillus and E. coli) and produced SCFA (Short Chain Fatty Acid) in Sprague Dawley Rats. Quantification of total microbes in the cecum performed with the molecular approach, namely FISH (fluorescence In situ hybridization) method. The rats were divided into 4 groups, each group 8 rats, group I were fed a standard (AIN ‘93), group II were fed a standard which dietary fiber was replaced by dried inulin (Inulin KR), group III were fed a standard which dietary fiber was replaced by inulin powder (inulin FM), and the fourth group were fed a standard which dietary fiber was replaced by commercial inulin (inulin SD). The results showed that lesser yam inulin was increased the Bifidobacterium from 7.46±0.5 log cfu/gr to 8.64±0.69 log cfu/g, and Lactobacillus from 7.71±0.72 log cfu/g to 9.05±0.94 log cfu/g. Lesser yam inulin was reduced E. coli from 7.70±0.79 log cfu/g to 6.99±0.33 log cfu/g. Lesser yam inulin was increased the production of SCFA (acetate, propionate and butyrate). This result indicated that the lesser yam inulin can be controlled colonic micro biota Sprague Dawley (SD) rats, so it can be used as a prebiotics.