The colonic epithelium harbors a large number of endocrine cells, but little is known about the endocrine functions of the colon. However, the high density of GLP-1 and PYY secreting L-cells is of great interest because of the potential anti-diabetic and anti-obesity effects of GLP-1 and PYY. Short chain fatty acids (SCFAs) produced by local bacterial fermentation are suggested to activate the colonic free fatty acid receptors FFAR2 (GPR43) and FFAR3 (GPR41), stimulating the colonic L-cells. We used the isolated perfused rat colon as a model of colonic endocrine secretion and studied the effects of the predominant SCFAs formed: acetate, propionate and butyrate. We show that luminal and especially vascular infusion of acetate and butyrate significantly increases colonic GLP-1 secretion, and to a minor extent also PYY secretion, but only after enhancement of intracellular cAMP. Propionate neither affected GLP-1 nor PYY secretion whether administered luminally or vascularly. A FFAR2 and FFAR3 specific agonist (CFMB/AR420626) had no effect on colonic GLP-1 output, and a FFAR3 antagonist (AR399519) did not decrease the SCFA-induced GLP-1 response. However, the voltage-gated Ca2+-channel blocker nifedipine, the KATP-channel opener diazoxide and the ATP synthesis inhibitor 2,4-dinitrophenol completely abolished the responses. FFAR2 receptor studies confirmed low-potent partial agonism of acetate, propionate and butyrate, compared to CFMB which is a full agonist with around 750-fold higher potency than the SCFAs. In conclusion, SCFAs may increase colonic GLP-1/PYY secretion, but FFAR2/FFAR3 do not seem to be involved. Rather, SCFAs are metabolized and appear to function as a colonocyte energy source.