Background: L-theanine is a natural bioactive amino acid present in tea (Camellia sinensis [L.] O. Kuntze) and consumed worldwide through tea decoction. Objective: The present work aims at evaluation of the insulin secretion and cytoprotective efficacy of L-theanine, a bioactive amino acid present in tea, on pancreatic β-cell line, RIN-m5F. Methods: RIN m5F cell were treated with L-theanine alone or prior to hydrogen peroxide (200 um) treatment and subsequently cell viability, cellular morphology, insulin secretion and insulin gene expression were analyzed. Results: Studies have shown that L-theanine dose dependently (0–300 μm) increases the β-cell mass as well as increases insulin production by RIN-m5F cell. It was also observed that pretreatment of the cell with L-theanine partially protected the oxidative stress of β-cells that were evident from cell viability, cellular morphology, and restoration of insulin-secreting ability. Conclusion: Results suggest that L theanine is an insulinotropic agent as well as effective in giving partial protection to pancreatic β-cells in oxidatively stressed condition. L-theanine can be used in the prevention or treatment of diabetes.