AIMS: The sphingolipid metabolite sphingosine 1‑phosphate (S1P) has emerged as a potential cardioprotective molecule against ischemic heart disease. Moreover, S1P triggers mobilization and homing of bone marrow-derived stem/progenitor cells into the damaged heart. However, it remains elusive whether S1P promotes mesenchymal stem cells (MSCs)-mediated cardioprotection against ischemic heart diseases. MAIN METHODS: Adipose tissue-derived MSCs (AT-MSCs) were obtained from GFP transgenic mice or C57BL/6J. Myocardial infarction (MI) was induced in C57BL/6J mice by ligation of the left anterior descending coronary artery (LAD). Subsequently, S1P-treated AT-MSCs or vehicle-treated AT-MSCs were intravenously administered for 24 h after induction of MI or sham procedure. KEY FINDINGS: Pre-conditioning with S1P significantly enhanced the migratory and anti-apoptotic efficacies of AT-MSCs. In MI-induced mice, intravenous administration of S1P-treated AT-MSCs significantly augmented their homing and engraftment in ischemic area. Besides, AT-MSCs with S1P pre-treatment exhibited enhanced potencies to inhibit cardiomyocyte apoptosis and fibrosis, and stimulate angiogenesis and preserve cardiac function. Mechanistic studies revealed that S1P promoted AT-MSCs migration through activation of ERK1/2-MMP-9, and protected AT-MSCs against apoptosis via Akt activation. Further, S1P activated the ERK1/2 and Akt via S1P receptor 2 (S1PR2), but not through S1PR1. S1PR2 knockdown by siRNA, however, significantly attenuated S1P-mediated AT-MSCs migration and anti-apoptosis. SIGNIFICANCE: The findings of the present study revealed the protective efficacies of S1P pretreatment on the survival/retention and cardioprotection of engrafted MSCs. Pre-conditioning of donor MSCs with S1P is an effective strategy to promote the therapeutic potential of MSCs for ischemic heart diseases.