To explore the effect of vitamin B1 on Diabetic Peripheral Neuropathy (DPN) and its mechanism for providing a theoretical basis for clinical prevention and effective treatment of diabetic neuropathy. 40 SD male rats were randomly divided into four groups (n=10/group) and they were normal control (NC) group, Diabetes Mellitus (DM) group, diabetic peripheral neuropathy (DPN) group and vitamin B1 (VB1) group. A rat model of diabetes was induced in DM, DPN and VB1 group rats by intra-peritoneal injecting a freshly prepared streptozotocin, and vitamin B1 was only given rats in VB1 group. Then electrochemical methods were used to detect the level of serum vitamin B1, while spectrophotometry was applied to determine the content of acetyl cholinesterase in sciatic nerve tissues, changes in inflammatory factor NF-κB could be presented using immunohistochemistry, the morphology of never tissues was observed under Transmission Electron Microscope (TEM). Compared with NC group, DM and DPN group showed decreased serum vitamin B1 and acetyl cholinesterase level in sciatic nerve tissues, but up-regulated NF-κB expression (P<0.05). Compared with DPN group, after vitamin B1 supplementation, the level of acetyl cholinesterase in sciatic nerve tissues was significantly increased, but NF-κB expression was significantly down-regulated in VB1 group (P<0.05). Electron microscope showed that rats with DPN presented uneven thickness of sciatic nerve myelin, partial lamellar separation in bent and twisted state, and some lamellar growth into axon to make the axon thin, as well as organelles degeneration, chromatin condensation, incomplete nuclear envelope in Schwann cells. At 12th week after vitamin B1 supplementation, the uneven thickness of sciatic nerve myelin and lamellar separation ameliorated; meanwhile, Schwann cells presented normal organelles basically and chromatin condensation. Taken together, vitamin B1 has some preventive and improving effects on DPN, and this effect may be achieved by changing acetyl cholinesterase content and NF-κB expression in sciatic nerve tissues.