The expression of short chain fatty acid receptors FFA2 and FFA3 in pancreatic islets raised interest in using them as drug targets for treating hyperglycemia in humans. This study aims to examine the efficacy of synthetic FFA2- and FFA3-ligands to modulate glucose-stimulated insulin secretion (GSIS) in human pseudoislets which display intact glucose responsiveness. The FFA2-agonists 4-CMTB and TUG-1375 inhibited GSIS, an effect reversed by the FFA2-antagonist CATPB. GSIS itself was not augmented by CATPB. The FFA3-agonists FHQC and 1-MCPC did not affect GSIS in human pseudoislets. For further drug evaluation we used mouse islets. The CATPB-sensitive inhibitory effect of 100 µM 4-CMTB on GSIS was recapitulated. The inhibition was partially sensitive to the Gi/o-protein inhibitor pertussis toxin. A previously described FFA2-dependent increase of GSIS was observed with lower concentrations of 4-CMTB (10 and 30 µM). The stimulatory effect of 4-CMTB on secretion was prevented by the Gq-protein inhibitor FR900359. As in human pseudoislets, in mouse islets relative mRNA levels were FFAR2 > FFAR3 and FFA3-agonists did not affect GSIS. The FFA3-agonists, however, inhibited GSIS in a pertussis toxin-sensitive manner in INS-1E cells and this correlated with relative mRNA levels of Ffar3 > > Ffar2. Thus, in humans, when FFA2-activation impedes GSIS, FFA2-antagonism may reduce glycemia.