Prolonged in vitro exposure to high glucose has been shown to induce a decrease in pancreatic B-cell sensitivity to glucose stimulation. This experiment was designed to study whether nicotinamide affects the B-cells' response to regulatory stimuli of glucose after prolonged culture with high glucose. Neonatal pig pancreatic islet-like cell clusters (ICCs) formed from single cells were embedded in agarose-gel. Some of them were maintained in RPMI 1640 containing a high glucose concentration (16.7 mM) and 10% fetal bovine serum (FBS) with or without nicotinamide. Then, the embedded-ICCs were stimulated by glucose at concentrations of 5.5 mM and 16.7 mM. After the prolonged culture with high glucose, the ICCs showed no response to acute glucose stimulation. There was a significant increase in glucose-mediated insulin secretion when the ICCs were maintained with the medium containing nicotinamide. We conclude that nicotinamide could protect B-cell desensitization to glucose after prolonged exposure to high glucose.