G-protein coupled receptor 55 (GPR55)is an orphan G-protein coupled receptor, which is activated by endocannabinoids and lipid transmitters. Recently, GPR55 was shown to play a role in glucose and energy homeostasis, and insulin secretion is essential to maintain glucose homeostasis in the body. In Type 2 Diabetes Mellitus (T2DM), chronic insulin resistance and a progressive decline in β-cell function result in β-cell dysfunction, this leads to defect in insulin secretion, which is the key process in the development and progression of T2DM. GPR55 agonists were shown to increase insulin secretion, however the underlying mechanisms were not fully understood. Therefore the aim of the present study was to examine the effects of potent GPR55 agonists, O-1602 and abnormal cannabidiol (Abn-CBD), on glucose-induced insulin secretion in a mouse pancreatic β-cell line, MIN6, and the underlying mechanisms with a focus on intracellular calcium (Ca). Our results demonstrated that O-1602 and Abn-CBD increased glucose-induced insulin secretion in MIN6 cells, which was abolished by a PLC inhibitor, U73122. Glucose-induced Ca transients were enhanced by O-1602 and Abn-CBD, and this was significantly reduced by U73122 and inositol trisphosphate (IP)receptor inhibitors, 2-aminoethoxydiphenyl borate (2-APB)and xestospongin C, as well as by Y-27632, a Rho-associated protein kinase (ROCK)inhibitor. Interestingly, O-1602 and Abn-CBD could directly induce intracellular Ca transients through IP-mediated Ca release. In conclusion, GPR55 agonists increased insulin secretion through calcium mobilisation from IP-sensitive ER stores in β-cells.