Diabetic peripheral neuropathy (DPN), a common disorder in diabetes, is associated with severe microenvironment imbalance due to immunometabolic stress. However, the underlying mechanistic drivers remain unclear. Here, we generate a single-cell atlas of human peripheral nerves and identify cell-specific transcriptional changes in DPN as well as aberrant amplification of mast cells. Using streptozotocin-induced mouse diabetes models, we further find that glucose uptake mediated by GLUT3 in high-glucose (HG) diabetic milieu upregulates ERK1/2 phosphorylation in mouse mast cells. Sustained HG stimulation also induces aberrant mTOR hyperactivity, resulting in endoplasmic reticulum stress and mitochondrial oxidative stress, thereby impairing mitochondrial functions of mast cells. Dysregulated mast cells then degranulate and release histamine, tryptase and inflammatory factors into neural microenvironment to cause neuropathy in diabetic mice. Lastly, mice with mast cell deficiency are protected from the immune imbalance in nerves and progression of neuropathy. Our findings thus implicate dysregulated activation of mast cells as a potential driver in the progression of DPN.