Overactivated cyclin-dependent kinase 5 (Cdk5) induces Golgi fragmentation, which interrupts the processing and trafficking of secretory cargo and subsequently synaptic plasticity and synaptogenesis, and even leads to neuronal cell death. Cdk5 overactivation and subsequent Golgi fragmentation are involved in many neurodegenerative diseases. However, whether isoflurane-induced neurotoxicity is relevant to aberrant Cdk5 activation and subsequent Golgi fragmentation remains unknown. In the present study, we explored the underlying molecular mechanisms of isoflurane-induced neurotoxicity in primary cultured hippocampal neurons. After treatment with 2% isoflurane for 6 h, immunofluorescence staining and transmission electron microscopy were used to examine the Golgi structure. Neuronal viability was evaluated using the 3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and TUNEL staining. Cdk5 activity was assessed using histone H1 as a substrate. Our results showed that Cdk5 activity and the number of fragmented Golgi increased significantly after isoflurane exposure. This was accompanied by an increase in neuronal death. Meanwhile, pharmacological inhibition of Cdk5 activity by 8 µM roscovitine alleviated isoflurane-induced Golgi fragmentation and neurotoxicity. Cumulatively, this study shows that aberrant Cdk5 activation-induced Golgi fragmentation is relevant to isoflurane neurotoxicity and indicates that a Cdk5 inhibitor may be a potential therapeutic candidate for the prevention of isoflurane-induced neurotoxicity. Video abstract: http://links.lww.com/WNR/A445.