Sphingosine kinase 2 (SPK2) and autophagy are both involved in cerebral preconditioning, but whether preconditioning-induced SPK2 upregulation and autophagy activation are linked mechanistically remains to be elucidated.In this study, we used in vitro and in vivo models to explore the role of SPK2-mediated autophagy in isoflurane (ISO) and hypoxic preconditioning (HP).In primary cultured mouse cortical neurons, both ISO and HP induced autophagy, as evidenced by an elevated LC3Ⅱ/LC3Ⅰ ratio and p62 downregulation.ISO and HP protected against subsequent oxygen glucose deprivation (OGD) or glutamate (Glu) injury, while pretreatment with autophagy inhibitors (3-MA or KU55933) abolished preconditioning-induced tolerance.Pretreatment with SPK2 inhibitors (ABC294640 and SK1-Ⅱ) prevented preconditioning-induced autophagy.ISO also induced autophagy in the cortex of C57 mice as shown by western blots for LC3 and p62, immunoflurescence/immunohistochemistry of LC3 and electron microscopy.ISO induced autophagy in mice lacking the SPK1 isoform (SPKl-/-), but not in SPK2-/-mice.S1P and the S1P receptor agonist FTY720 did not protect against OGD in cultured neurons and did not alter the expression of LC3 and p62, suggesting that SPK2-mediated autopbagy and protections are not S1Pdependent.Beclin1 knockdown abolished preconditioning-induced autophagy activation, but SPK2 inhibitors abolished ISO-induced disruption of the Beclin1/Bcl-2 association.Taken together, these results strongly indicate that autophagy is involved in isoflurane preconditioning both in vivo and in vitro and that SPK2 contributes to preconditioning-induced autophagy, possibly by disrupting the Beclin 1/Bcl-2 interaction.