In response to the increased insulin resistance during pregnancy, pancreatic islets undergo not only an increase in β cell proliferation but also an increase in glucose stimulated insulin secretion (GSIS). Although several mechanisms for the increase of cell proliferation during pregnancy have been reported, the mechanism of the increased GSIS has yet to be elucidated. Here we explored the role of 5-HT in the regulation of GSIS by analyzing Htr3a KO mice. Htr3a KO mice showed impaired glucose tolerance during pregnancy in spite of normal increase of cell mass. GSIS was markedly increased in the pancreatic islets isolated from pregnant mice but not in the islets isolated from pregnant Htr3a KO mice. Activation of 5-HT3 receptor (Htr3) during pregnancy induced increased uptake of Ca2+ in response to glucose stimulation, which resulted in increased insulin exocytosis. Electrophysiological studies showed that activation of Htr3 induced a depolarizing shift of resting membrane potential in β cells, which decreased glucose threshold for insulin secretion. Thus, our data indicated that Htr3 signaling, in a paracrine/autocrine manner, plays an essential role in the increase of GSIS during pregnancy.