Endothelial cell dysfunction plays an essential role in the process of cardiac ischemia-reperfusion (I/R) injury. Mitochondria damage, which can trigger inflammasome activation and subsequent pyroptosis, perturbs endothelial homeostasis, leading to aggravated cardiac I/R injury. Sphingosine 1-phosphate (S1P), a bioactive lipid molecule, exerts multifaceted effect on I/R injury via its different S1P receptors. However, the effect of EC-expressing S1P receptors on endothelial dysfunction, mitochondrial damage-induced inflammasome activation and consequent pyroptosis during cardiac I/R injury remain unclear. Our findings suggest a pivotal role of EC-expressing S1PR2 to control EC mitochondrial homeostasis and demonstrate that S1PR2-meidated mitochondrial dysfunction can trigger inflammasome activation and pyroptosis in ECs, which significantly influences inflammatory responses and heart injuries following I/R. Overall design: We provided an RNA-seq analysis on heart tissues of WT and VECre-S1pr2-/- mouse after cardiac ischemia-reperfusion.