Low serum bicarbonate is closely related to type 2 diabetes mellitus. However, the precise role of bicarbonate on glucose homeostasis and insulin secretion remains unknown. In this study, we investigated the effects of bicarbonate concentration on pancreatic 0-cells. It was observed that the high bicarbonate concentration of the cell culture medium significantly increased the glucose-induced insulin secretion (GSIS) levels in mouse islets, MIN6, and the INS-1E 0 cells. MIN6 cells presented an impaired GSIS; the cells produced a lower bicarbonate concentration when co-cultured with Capan-1 than when with CFPAC-1. NBCe1, a major bicarbonate transporter was observed to block the increasing insulin secretions, which were promoted by a high concentration of bicarbonate. In addition, higher extracellular bicarbonate concentration significantly increased the intracellular cAMP level, pHi, and calcium concentration with a 16.7 mM of glucose stimulation. Further study demonstrated that a low concentration of extracellular bicarbonate significantly impaired the functioning of pancreatic 0 cells by reducing coupling Ca2+ influx, whose process may be modulated by NBCe1. Taken together, our results conclude that bicarbonate may serve as a novel target in diabetes prevention-related research.