Vascular endothelial growth factor B (VEGFB) plays a crucial role in glucolipid metabolism and is highly associated with type 2 diabetes mellitus (T2DM). The role of VEGFB in the insulin secretion of β cells remains unverified. Thus, this study aims to discuss the effect of VEGFB on regulating insulin secretion in T2DM development, and its underlying mechanism. A high-fat diet and streptozocin were used for inducing T2DM in mice model, and VEGFB gene in islet cells of T2DM mice was knocked out by CRISPR Cas9 and overexpressed by Adeno-Associated Virus (AAV) injection. The effect of VEGFB and its underlying mechanism was assessed by light microscope, electron microscope, fluorescence confocal microscope, enzyme-linked immunosorbent assay, mass spectrometer, and western blot. The decrement of insulin secretion in islet β cell of T2DM mice are aggravated and blood glucose remains at a high level after VEGFB deletion. However, glucose tolerance and insulin sensitivity of T2DM mice were improved after the AAV-VEGFB186 injection. VEGFB knockout or overexpression can inhibit or activate PLCγ/IP3R in a VEGFR1-dependent manner. Then, the change of PLCγ/IP3R caused by VEGFB/VEGFR1 will alter the expression of key factors on the Ca2+/CaMK2 signal pathway such as PPP3CA. Moreover, VEGFB can cause altered insulin secretion by changing the calcium concentration in β cells of T2DM mice. These findings indicated that VEGFB activated the Ca2+/CaMK2 pathway via VEGFR1-PLCγ and IP3R pathway to regulate insulin secretion, which provides new insight into the regulatory mechanism of abnormal insulin secretion in T2DM.