High glucose induces Schwann cells death and neurotoxicity. Formononetin was originally found in Astragalus membranaceus and showed anti-tumor and anti-neuroinflammation properties. The aim of this study is to explore the molecular mechanism underlying the neuroprotective effects of formononetin and identify its direct protein target. The effects of formononetin on oxidative stress and mitochondrial dysfunction in Schwann cells induced by high glucose were investigated. High glucose treatment significantly induced oxidative stress, mitochondrial dysfunction and apoptosis in Schwann cells, while these effects were partially or completely prevented by co-treatment with formononetin. Mechanistically, we found that SIRT3/PGC-1α/SOD2 pathway was activated by formononetin under high glucose conditions as evidenced by western blotting. Knockdown of SIRT3 by siRNA delivery reversed the protective effects of formononetin on high glucose-induced Schwann cells injury and changes in expression profile of SIRT3 downstream target genes. Molecular docking, thermal shift assay and surface plasmon resonance assay revealed a direct binding between formononetin and SIRT3. Taken together, we identified a novel SIRT3 activator formononetin and revealed its beneficial effects on high glucose-induced neurotoxicity, suggesting that targeting SIRT3 in Schwann cells may be a new approach for treatment of peripheral nerve regeneration related diseases such as diabetic peripheral neuropathy.