In preparation for the metabolic demands of pregnancy, β cells in the maternal pancreatic islets increase both in number and in glucose stimulated insulin secretion (GSIS) per cell. Mechanisms have been proposed for the increased β cell mass, but not for the increased GSIS. Because serotonin production increases dramatically during pregnancy, we tested whether flux through the ionotropic serotonin receptor Htr3 impacts GSIS during pregnancy. Pregnant Htr3a^-/- mice exhibited impaired glucose tolerance despite normally increased β cell mass, and their islets lacked the increase in GSIS seen in islets from pregnant wildtype mice. Electrophysiological studies showed that activation of Htr3 decreased the resting membrane potential in β cells, which increased Ca2+ uptake and insulin exocytosis in response to glucose. Thus, our data indicate that serotonin, acting in a paracrine/autocrine manner through Htr3, lowers the β cell threshold for glucose and plays an essential role in the increased GSIS of pregnancy.