A large number of glucagon-like-peptide 1 (GLP-1) and peptide-YY (PYY) producing L-cells are located in the colon, but little is known about their contribution to whole body metabolism. Since bile acids (BAs) increase GLP-1 and PYY release, and since BAs spill over from the ileum to the colon, we decided to investigate the ability of BAs to stimulate colonic GLP-1 and PYY secretion. Using isolated perfused rat/mouse colon as well as stimulation of the rat colon in vivo, we demonstrate that BAs significantly enhance secretion of GLP-1 and PYY from the colon with average increases of 3.5 and 2.9-fold, respectively. Furthermore, we find that responses depend on BA absorption followed by basolateral activation of the BA-receptor, TGR5. Surprisingly, the apical sodium-dependent-bile-acid-transporter (IBAT), which serves to absorb conjugated BAs, was not required for colonic conjugated BA absorption or conjugated BA-induced peptide secretion. In conclusion, we demonstrate that BAs represent a major physiological stimulus for colonic L-cell secretion.