To investigate the mechanisms of the axonopathy induced by 2,5-hexanedione (2,5-HD), male Wistar rats were administered at a dosage of 400 mg/kg/day 2,5-HD (five times per week). The rats produced a slightly, moderately, or severely abnormal neurological changes, respectively, after 2, 4, or 8 weeks of treatment. The cerebrums were Triton-extracted and ultracentrifuged to yield a pellet fraction and a corresponding supernatant fraction. The relative levels of six cytoskeletal proteins (NF-L, NF-M, NF-H, alpha-tubulin, beta-tubulin, and beta-actin) in both fractions were determined by immunoblotting. The results showed that NFs content in HD-treated rats demonstrated a progressive decline as the intoxication of HD continued. As for microtubule proteins, the levels of alpha-tubulin and beta-tubulin demonstrated some inconsistent changes. The content of alpha-tubulin kept unchangeable, while the content of beta-tubulin increased significantly at the late stage of HD exposure. Furthermore, the content of beta-actin in both fractions remained unaffected throughout the study. These findings suggest that HD intoxication resulted in a progressive decline of NFs, which was highly correlated with the development of HD-induced neuropathy.