Congenital hyperinsulinism (HI) is a genetic disorder in which pancreatic beta cell insulin secretion is excessive and results in hypoglycemia that can cause brain damage or death without treatment. Most patients with loss-of-function mutations in ABCC8 and KCNJ11, the genes encoding the beta cell ATP sensitive potassium channel (KATP), are unresponsive to diazoxide, the only FDA approved medical therapy, and require a pancreatectomy. The glucagon-like peptide 1 receptor (GLP-1R) antagonist exendin-(9-39) is an effective therapeutic agent to inhibit insulin secretion in both congenital and acquired hyperinsulinism. Previously, we identified a highly potent antagonist antibody, TB-001-003, which was derived from our synthetic antibody Variant Libraries that were designed to target G protein-coupled receptors. Here, we designed a combinatorial variant antibody library to optimize the activity of TB-001-003 against GLP-1R and performed phage-display on cells overexpressing GLP-1R. One antagonist, TB-222-023, is more potent than exendin-(9-39), also known as Avexitide. TB-222-023 effectively decreased insulin secretion in primary isolated pancreatic islets from a mouse model of hyperinsulinism, Sur1-/- mice, and in islets from an infant with HI as well as increased plasma glucose and decreased the insulin to glucose ratio in Sur1-/- mice. These findings demonstrate that targeting GLP-1R with an antibody antagonist is an effective and innovative strategy for treatment of hyperinsulinism.