Glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are anti-diabetes/obesity hormones secreted from the gut after meal ingestion. We have shown that dietary resistant starch increased GLP-1 and PYY secretion, but the mechanism remains unknown. Resistant starch (RS) is a fermentable fiber, which lowers the glycemic index of the diet and liberates short chain fatty acids (SCFAs) through fermentation in the gut. This study investigates the two possible mechanisms by which RS stimulates GLP-1 and PYY secretion: the effect of meal or glycemic index and the effect of fermentation. First, because GLP-1 and PYY secretions are stimulated by nutrient availability in the gut, the timing of blood sample collections could influence the outcome when comparing two diets that have different glycemic indexes. Thus, we examined GLP-1 and PYY plasma levels at various time points over a 24 hour period in RS fed rats. Secondly, we tested proglucagon (a precursor to GLP-1) and PYY gene expression patterns in specific areas of the gut for RS fed rats and in the enteroendocrine cell line following exposure to SCFAs in vitro. Results: 1) Dietary resistant starch stimulates GLP-1 and PYY secretion in a substantial daylong manner, independent of meal effect or changes in dietary glycemia. 2) Fermentation and the liberation of SCFAs in the lower gut are associated with increased proglucagon and PYY gene expressions. Lastly, RS fed diabetic mice had improved glucose tolerance, an indicator of increased active forms of GLP-1 and PYY. Conclusion: Fermentation of RS is most likely the primary mechanism for increased endogenous secretions of total GLP-1 and PYY in rodents. Thus, any factor that affects fermentation should be considered when using dietary fermentable fiber to stimulate GLP-1 and PYY secretion.