Purpose: To evaluate the protective effect, effective time window, and mechanism of protection of sevoflurane post-conditioning and late sevoflurane post-conditioning on heart muscle ischemiareperfusion (I/R) lesion in isolated rat heart.Methods: Langendorff isolated perfusion model was established in Sprague-Dawley (SD) rats. After 15 min of stabilization, the rats were assigned to 10 groups, based on the treatments given: control group (TTC), I/R group, sevoflurane post-processing group (SpostC), 0.5 min-, 1 min-, 5 min-, 10 min-, 20 min-, 30 min- delayed sevoflurane post-processing group (S0.5, S1, S3, S5, S10, and S20). The levels of mitochondrial membrane permeability, area of myocardial infarction, myocardial apoptosis, and p-GSK3 beta level were determined.Results: Myocardial infarction areas in Spost, S0.5, S1, S3, S5, S10 and S20 groups were significantly than in I/R group (p < 0.05), while expression levels of myocardial p-Akt were significantly higher in Spost, S0.5, S1, S3, S5, S10, S20, and S30 groups than in I/R group (p < 0.05). GSK-3 phosphorylation level was significantly higher in Spost, S1, S10, S20, and S30 groups than in I/R group (p < 0.05). Expression of p-GSK-3β in S30 group decreased, relative to that in Spost group (p < 0.05).Conclusion: Sevoflurane post-conditioning and delayed sevoflurane post-conditioning mitigates I/R lesions in isolated rat hearts. The protective effect against ischemia-reperfusion injury is related to the levels of phosphorylation of Akt and GSK-3β. Thus, this treatment approach may be useful in the management of cardiac ischemia-reperfusion injury.