The glymphatic system is a recently discovered glial-dependent macroscopic interstitial waste clearance system that promotes the efficient elimination of soluble proteins and metabolites from the central nervous system. Its anatomic foundation is the astrocytes and aquaporin-4 (AQP4) water channels on the endfeet of astrocytes. The aim of this study is to evaluate the plasticity of the spinal glymphatic system in male SD rats with painful diabetic neuropathy (PDN) induced by type 2 diabetes mellitus. PDN rats were modeled under a high-fat and high-glucose diet with a low dose of streptozotocin. MRI was applied to observe the infiltration and clearance of contrast to indicate the functional variability of the glymphatic system at the spinal cord level. The paw withdrawal threshold was used to represent mechanical allodynia. The numerical change of glial fibrillary acidic protein (GFAP) positive astrocytes was assessed and the polarity reversal of AQP4 protein was measured by immunofluorescence. As a result, deceased contrast infiltration and clearance, enhanced mechanical allodynia, increased number of GFAP positive astrocytes, and reversed polarity of AQP4 protein were found in the PDN rats. The above molecular level changes may contribute to the impairment of the spinal glymphatic system in PDN rats. This study revealed the molecular and functional variations of the spinal glymphatic system in PDN rats and for the first time indicated that there might be a correlation between the impaired spinal glymphatic system and PDN rats.