The role of 5-HT in maintaining glucose homeostasis during metabolic stress and inhibiting glucagon secretion is well documented, however, its effect on α cell identity remained unclear. In this study, we demonstrated that 5-HT suppressed the expression of α cell markers, such as Arx and Gcg, while enhancing the expression of β cell markers in mouse pancreatic α cell lines. We further found that treatment with 5-HT significantly increased the percentage of Gcg+Ins+ and Gcg+Nkx6.1+ cells in isolated human and mouse islets. Using pancreatic α cell lineage-tracing Gcgcre+; tdTomato/tdTomato mice, we observed that 5-HT treatment significantly reduced random blood glucose levels and increased tdTomato+Ins+, Gcg+Ins+ and Gcg+Nkx6.1+ cells in a high fat diet and streptozotocin (HFD + STZ) induced diabetes model. Additionally, in situ detection of 5-HT production in the human pancreas revealed a reduction of 5-HT expression in β cells of human T2D patients. These findings suggest that 5-HT treatment induces the transdifferentiation of α to β cells, potentially contributing to the recovery of β cell mass in T2D.