Germinated barley foodstuff (GBF) derived from the aleurone and scutellum fractions of germinated barley mainly consists of low-lignified hemicellulose and glutamine-rich protein. GBF improves the proliferation of intestinal epithelial cells and defecation, through the bacterial production of short chain fatty acids (SCFA), especially butyrate. In this study we investigated the mechanism of production of butyrate by microflora in humans and in vitro. Daily administration of 9 g GBF for 14 successive days significantly increased fecal butyrate content. Fecal Bifidobacterium and Eubacterium were also significantly increased by GBF administration in healthy volunteers. Ten anaerobic micro-organisms selected from intestinal microflora were cultured in vitro in the medium containing GBF as a sole carbon source (GBF medium). After a 3-day incubation, 7 strains (Bifidobacterium breve, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus casei subsp. casei, Bacteroides ovatus, Clostridium butyricum, and Eubacterium limosum) lowered the medium pH producing SCFA. Eubacterium grown together with Bifidobacterium in GBF medium efficiently produced butyrate. On the other hand, GBF changed the intestinal microflora and increased probiotics such as Bifidobacterium in the intestinal tract. As a result, butyrate was produced by the mutual action of Eubacterium and Bifidobacterium. This butyrate is considered to enhance the proliferation of colonic epithelial cells.