Histamine receptor H₃ (H₃R), a G_i/o-coupled receptor, is dominantly expressed in the central nervous system and regulates neurotransmitter release. Our previous study showed that H₃R was also expressed in the pancreatic β cells and had an inhibitory effect on glucose-induced insulin secretion from MIN6 cells, a cell line from mouse pancreatic β cells. However, the in vivo roles of H₃R on β cells in the regulation of plasma insulin level are still unknown. In the present study, we generated and phenotyped β cell specific H₃R knockout mice (cKO mice) to elucidate the importance of H₃R for glucose homeostasis. Blood glucose testing showed that H₃R deletion from β cells resulted in the lower glucose levels after glucose challenge due to higher insulin secretion. Glucose-induced insulin secretion from isolated cKO islets was higher than that from control islets. These data demonstrated that disruption of H₃R led to higher insulin secretion in vivo. Immunohistochemical analysis showed that the number of Ki67-positive β cells was augmented in cKO islets. Morphometric analysis revealed the increased number and size of islets in cKO pancreas. These data indicated that proliferation of β cells was enhanced by H₃R deletion. In conclusion, H₃R in pancreatic β cells has negative impact on insulin secretion and their proliferation.