Butyrate was shown to have a preventive effect on colon cancer in vivo. Germinated barley foodstuff (GBF) was in a prebiotic stage and had the potency to attenuate mucosal inflammation and to increase fecal butyrate production in colitis. This study aimed to determine whether the GBF treatment in a colon cancer model had the potency to suppress colon cancer. After a pre-feeding of either a control or a GBF diet for two weeks, male F344 rats received subcutaneous injections of azoxymethane twice, at a dose level of 15 mg/kg body weight. The injections were administered once a week for 2 weeks (n=10/group). Four weeks after that, the number of aberrant crypt foci (ACF) and heat shock protein (HSP) 25-positive cells in colonic mucosa were observed histologically. The mRNA level of slc5a8 was evaluated by in situ hybridization. Colonic mucosal beta-catenin was determined by Western blotting. Cecal short chain fatty acids, beta-glucosidase and beta-glucuronidase were also determined. The results showed that GBF treatment significantly decreased the number of ACF and beta-catenin formations in the colonic mucosa. GBF significantly increased the production of slc5a8, which is a tumor suppressor gene, as well as the cecal butyrate content and beta-glucosidase activity. beta-glucuronidase activity remained at the same level in GBF and control subjects. The number of HSP25-positive cells in GBF was higher than that in the control group, although it did not reach significant difference. In conclusion, GBF showed anti-tumorigenicity in the AOM rat model. Changes in the colonic environment featured through the increase of butyrate production were found. Although a more detailed study is required, this study showed the promising anti-neoplastic effects of prebiotic treatment.