The G_q-coupled 5-hydroxytryptamine 2B (5-HT_2B) receptor is known to regulate the proliferation of islet beta cells during pregnancy. However, the role of serotonin in the control of insulin release is still controversial. The aim of the present study was to explore the role of the 5-HT_2B receptor in the regulation of insulin secretion in mouse and human islets, as well as in clonal INS-1(832/13) cells. Expression of HTR2B mRNA and 5-HT_2B protein was examined with quantitative real-time PCR, RNA sequencing and immunohistochemistry. α-Methyl serotonin maleate salt (AMS), a serotonin receptor agonist, was employed for robust 5-HT_2B receptor activation. Htr2b was silenced with small interfering RNA in INS-1(832/13) cells. Insulin secretion, Ca²⁺ response and oxygen consumption rate were determined. Immunohistochemistry revealed that 5-HT_2B is expressed in human and mouse islet beta cells. Activation of 5-HT_2B receptors by AMS enhanced glucose-stimulated insulin secretion (GSIS) in human and mouse islets as well as in INS-1(832/13) cells. Silencing Htr2b in INS-1(832/13) cells led to a 30% reduction in GSIS. 5-HT_2B receptor activation produced robust, regular and sustained Ca²⁺ oscillations in mouse islets with an increase in both peak distance (period) and time in the active phase as compared with control. Enhanced insulin secretion and Ca²⁺ changes induced by AMS coincided with an increase in oxygen consumption in INS-1(832/13) cells. Activation of 5-HT_2B receptors stimulates GSIS in beta cells by triggering downstream changes in cellular Ca²⁺ flux that enhance mitochondrial metabolism. Our findings suggest that serotonin and the 5-HT_2B receptor stimulate insulin release.