Aim To investigate the effect of alfentanil on myocardial ischemia-reperfusion injury (MIRI) in rats and its regulatory mechanism on the sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P) signaling pathway during this process. Methods SPF grade SD male rats were randomly divided into sham surgery group, model group, positive drug group (compound salvia miltiorrhiza group), low dose alfentanil group, high dose alfentanil group, and high alfentanil+SphK1 agonist group (alfentanil+PMA group), with 20 rats in each group. Except the sham operation group, the MIRI model was reproduced by ligating the left anterior descending coronary artery and reperfusion. The activities of serum lactate dehydrogenase (LDH), creatine kinase (CK) and aspartate aminotransferase (AST) were detected by automatic biochemical analyzer; TTC was applied to detect the size of myocardial infarction in rats; HE staining was applied to observe the morphological characteristics of rat myocardial tissue; TUNEL staining was applied to detect myocardial cell apoptosis in rats; ELISA was applied to detect the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and S1P; kits were applied to detect content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) in myocardial tissue; Western blot was applied to detect the expression level of SphK1 protein in myocardial tissue. Results Compared with the sham surgery group, the pathological damage to the myocardial tissue of rats was severe in the model group, the activities of serum central muscle injury markers LDH, CK, and AST, myocardial infarction area, myocardial cell apoptosis rate, the levels of TNF-α, IL-6, IL-1β, MDA, S1P and the expression of SphK1 protein all increased, the activity of SOD decreased (P＜0.05). Compared with the model group, the myocardial tissue damage of rats was reduced in the positive drug group and the low and high dose alfentanil groups, the activities of serum central muscle injury markers LDH, CK, and AST, myocardial infarction area, myocardial cell apoptosis rate, the levels of TNF-α, IL-6, IL-1β, MDA, S1P and the expression of SphK1 protein all decreased, the activity of SOD increased (P＜0.05). The SphK1 agonist was able to reverse the impact of high-dose alfentanil on the above indicators (P＜0.05). Conclusion Alfentanil has protective effect on MIRI rats, and its mechanism may be related to the inhibition of SphK1/S1P signaling pathway.