Diabetic peripheral neuropathy (DPN) is a common complication of diabetes, which has yet no curable medication. Neuroinflammation and mitochondrial dysfunction are tightly linked to DPN pathology. G-protein-coupled receptor 40 (GPR40) is predominantly expressed in pancreatic β-cells, but also in spinal dorsal horn and dorsal root ganglion (DRG) neurons, regulating neuropathic pain. We previously have reported that vincamine (Vin), a monoterpenoid indole alkaloid extracted from Madagascar periwinkle, is a GPR40 agonist. In this study, we evaluated the therapeutic potential of Vin in ameliorating the DPN-like pathology in diabetic mice. Both STZ-induced type 1 (T1DM) and db/db type 2 diabetic (T2DM) mice were used to establish late-stage DPN model (DPN mice), which were administered Vin (30 mg·kg-1·d-1, i.p.) for 4 weeks. We showed that Vin administration did not lower blood glucose levels, but significantly ameliorated neurological dysfunctions in DPN mice. Vin administration improved the blood flow velocities and blood perfusion areas of foot pads and sciatic nerve tissues in DPN mice. We demonstrated that Vin administration protected against sciatic nerve myelin sheath injury and ameliorated foot skin intraepidermal nerve fiber (IENF) density impairment in DPN mice. Moreover, Vin suppressed NLRP3 inflammasome activation through either β-Arrestin2 or β-Arrestin2/IκBα/NF-κB signaling, improved mitochondrial dysfunction through CaMKKβ/AMPK/SIRT1/PGC-1α signaling and alleviated oxidative stress through Nrf2 signaling in the sciatic nerve tissues of DPN mice and LPS/ATP-treated RSC96 cells. All the above-mentioned beneficial effects of Vin were abolished by GPR40-specific knockdown in dorsal root ganglia and sciatic nerve tissues. Together, these results support that pharmacological activation of GPR40 as a promising therapeutic strategy for DPN and highlight the potential of Vin in the treatment of this disease. © 2023, The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.