Objective Previously, exchange protein directly activated by cAMP (Epac)2 and PKA were known to play a role in glucose-stimulated insulin secretion (GSIS) by pancreatic β-cells. The objective of current study is to investigate the role of Epac1, which is another isoform of Epac, in pancreatic β-cell biology, since Epac1 mRNA and protein are also expressed by β-cells. Methods The embryonic stem (ES) cells with heterozygous and homozygous deletion of the Epac1 gene were generated to create Epac1-deficient mice and to differentiate to β-cells, respectively. These in vivo and in vitro systems were used for defining the role of Epac1 in β-cell function and metabolism. Results The homozygous Epac1-knockout (Epac1-/-) mice developed impaired glucose tolerance and GSIS by β-cells with an abnormal cytoarchitecture of α- and β-cells distribution in islets with lower expression of Glut2 on the β-cell membrane. Moreover, Epac1-/- mice developed more severe hyperglycemia with increased β-cell apoptosis and insulitis than wild type (Epac1+/+) mice after multiple low-dose streptozotocin (MLDS; 40 mg/kg) treatment. Interestingly, Epac1-/- mice also showed metabolic defects, including increased respiratory exchange ratio and plasma triglyceride, and more severe diet-induced obesity with insulin resistance, which may contributed to β-cell dysfunction. Therefore, islet-like clusters generated from Epac1+/+ and Epac1-/- ES cells were used to further understand the direct role of Epac1 in β-cells. The β-cells derived from Epac1-/- ES cells showed insulin secretion defect, reduced expressions of Glut2 and Pdx1, and abolished GLP-1-stimulated PCNA induction. Conclusion The current study provides in vitro and in vivo evidence that Epac1 is important in glucose metabolism partly by regulating GSIS and Glut2 expression in β-cells, GLP-1-induced β-cell proliferation.