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Chronic exposure to n-hexane causes sensorimotor neuropathy, which is mediated by 2,5-hexanedione (HD), a toxic metabolite of n-hexane. Activation of the nucleotide-binding and oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3 (NLRP3) inflammasome is involved in multiple neurodegenerative diseases. However, whether the NLRP3 inflammasome contributes to HD-induced neurotoxicity remains unclear. In this study, the effects of HD on NLRP3 inflammasome activation and the underlying mechanisms were determined by using HD-treated rat and cell culture models. Increased NLRP3 expression, caspase-1 activation and interleukin-1β production were observed in both the brain and spinal cord of HD-treated rats. Double-immunofluorescence staining showed that ASC speck formation and caspase-1 expression were mainly localized in microglia. HD-induced activation of the NLRP3 inflammasome was further mirrored in BV2 microglial cells and was associated with NADPH oxidase activation. Interestingly, inhibition of NADPH oxidase by apocynin or specific siRNAs significantly mitigated HD-induced NLRP3 inflammasome activation. Furthermore, apocynin suppressed activation of the MAPK and NF-κB signaling pathways. Blocking activation of p38-MAPK and NF-κB significantly reduced HD-induced capase-1 activation and interleukin-1β maturation, indicating a critical role of NADPH oxidase and downstream MAPK and NF-κB pathways in regulating activation of NLRP3 inflammasome, in HD-treated microglia. Finally, we found that inhibition of microglial NLRP3 inflammasome and NADPH oxidase activation abrogated HD-induced microglial activation and neurodegeneration in both SHSY5Y neuronal cells and primary cortical neuron-glia cultures. Altogether, our findings suggest that NADPH oxidase-dependent activation of microglial NLRP3 inflammasome contributes to HD-induced neurotoxicity, providing novel insight into the mechanisms of this solvent-induced neuropathy.