Abstract: AIM:To investigate the difference of sphingosine-1-phosphate (S1P)-mediated protection against hypoxia/reoxygenation (H/R) induced cardiomyocyte injury between pre- and post-hypoxia treatment and the mechanism(s) involved. METHODS: Neonatal rat ventricular myocytes (NRVMs) were isolated from 1- to 2-day-old Sprague Dawley (SD) rats and were subjected to 9 h of hypoxia followed by 3 h of reoxygenation. After pre- or post-hypoxia treatment with 1 μmol/L S1P, cell viability was detected by MTT assay. LDH release and caspase-3 activity were detected by assay kits. AMPK, Akt and ERK1/2 phosphorylation levels and S1P lyase 1(SPL1) expression levels were analyzed by Western blot. RESULTS: In cultured NRVMs, pre-hypoxia treatment with 1 μmol/L S1P increased AMPK, Akt and ERK1/2 phosphorylation levels (all P<0.05) increased cell viability and reduced LDH release and caspase-3 activity after H/R (all P<0.01). However, post-hypoxia treatment with 1 μmol/L S1P failed to upregulate AMPK, Akt and ERK1/2 phosphorylation levels and lost its cardioprotective effects compared with those in H/R group. SPL1 expression level in NRVMs increased after hypoxia treatment (P<0.01). CONCLUSION: Pre-hypoxia treatment with S1P, but not post-hypoxia treatment, exerts strong protective effects against H/R injury in NRVMs by decreasing apoptosis. Pre-hypoxia treatment with S1P activates AMPK, Akt and ERK1/2 survival signaling pathway, whereas post-hypoxia treatment with S1P fails to achieve these effects. The mechanisms may be that hypoxia-induced SPL1 upregulation dismisses the protective role of S1P against H/R induced cardiomyocyte injury.