Isoflurane is a commonly used inhalational anesthetic that can induce neurotoxicity, while Dexmedetomidine (Dex) has significant neuroprotective effects. In our study, we explored the effects of Dex on isoflurane-induced neurotoxicity through the TLR2/NF-κB signaling pathway. Seven-day old neonatal Sprague-Dawley rats pretreated with 25, 50, 75 μg/kg Dex were exposed to 0.75% isoflurane for 6 h. Spatial learning and memory abilities were detected by Morris water maze test. Ultrastructure of hippocampal neurons, neuronal apoptosis, and the levels of TLR2/NF-κB signaling pathway-related factors were determined. Besides, TLR2 agonist Pam3CSK4 or NF-κB inhibitor BAY11-7082 was injected to further validate the involvement of TLR2/NF-κB signaling following Dex treatment. Consequently, we found isoflurane inhalation resulted in increased cell apoptosis, inflammation and TLR2/NF-κB signaling pathway activation, and decreased PSD95 expression and spatial learning and memory abilities. Dex led to decreased inflammation, improved neuronal structure and viability in rats as well as enhanced spatial learning and memory abilities of rats, and it inactivated the TLR2/NF-κB signaling pathway. Additionally, Pam3CSK4 injection reversed the protective effects of Dex on isoflurane-induced neurotoxicity. In conclusion, this study provided evidence that Dex could alleviate isoflurane-induced neurotoxicity through inhibition of the TLR2/NF-κB signaling pathway. The findings may offer novel insights for the clinical usage of anesthetics.