This study aims to determine whether caveolin-1 (Cav-1) participates in the process of diabetic neuropathic pain by directly regulating the expression of toll-like receptor 4 (TLR4) and the subsequent phosphorylation of N-methyl-D-aspartate receptor 2B subunit (NR2B) in the spinal cord. Male Sprague-Dawley rats (120-150 g) were continuously fed with high-fat and high-sugar diet for 8 weeks, and received a single low-dose of intraperitoneal streptozocin injection in preparation for the type-II diabetes model. Then, these rats were divided into five groups according to the level of blood glucose, and the mechanical withdrawal threshold and thermal withdrawal latency values. The pain thresholds were measured at 3, 7, and 14 days after animal grouping. Then, eight rats were randomly chosen from each group and killed. Lumbar segments 4-6 of the spinal cord were removed for western blot analysis and immunofluorescence assay. Cav-1 was persistently upregulated in the spinal cord after diabetic neuropathic pain in rats. The downregulation of Cav-1 through the subcutaneous injection of Cav-1 inhibitor daidzein ameliorated the pain hypersensitivity and TLR4 expression in the spinal cord in diabetic neuropathic pain (DNP) rats. Furthermore, it was found that Cav-1 directly bound with TLR4, and the subsequent phosphorylation of NR2B in the spinal cord contributed to the modulation of DNP. These findings suggest that Cav-1 plays a vital role in DNP processing at least in part by directly regulating the expression of TLR4, and through the subsequent phosphorylation of NR2B in the spinal cord.