A non-proteinaceous amino acid called GABA is well-known for its physiological uses and its role as an inhibitory neurotransmitter in the brain of mammals. Apart from its neurological function, GABA has been linked to blood pressure reduction and glucose balance modulation. Since β-cell destruction and decreased insulin secretion are hallmarks of diabetes mellitus, the natural production of GABA by pancreatic β-cells increases the possibility of its therapeutic role. The aim of this study was to produce, purify, and characterize GABA from glutamic acid and assess its potential as an antidiabetic, namely to impact β-cell proliferation. GABA was produced from glutamic acid using the enzyme glutamate decarboxylase (GAD). After that, the produced GABA was purified and characterized using different analytical methods. Furthermore, in vivo studies were carried out with diabetic mice to evaluate the effect of GABA treatment on β-cell proliferation and control of insulin levels. GABA treatment improved glycaemic control in diabetic mice by increasing β-cell proliferation and regulating glucagon and insulin production. These results demonstrate the potential of GABA as an antidiabetic drug and provide a safe substitute for traditional insulin injections. Further study is needed to understand its mechanism of action and investigate its therapeutic application in the treatment of diabetes. © 2025 Elsevier B.V., All rights reserved.