Accumulating evidence has confirmed that the expression of sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) is downregulated in heart failure and cardiac allograft rejection. Although many SERCA2a-related genes and proteins involved in the regulation of myocardial Ca2+ fluxes have been explored, its related metabolites remain poorly studied. Our main objective was to identify circulating SERCA2a-related metabolites altered in cardiac allograft rejection and to determine whether these could serve as non-invasive biomarkers. Sixty plasma samples from adult heart transplant were included in a metabolomic analysis. Sphingosine-1 phosphate (S1P), metabolite closely related with SERCA, were increased in patients with cardiac rejection (p < 0.0001). S1P discriminated between patients with and without rejection: normal grafts vs. all rejecting grafts (AUC = 0.911, p < 0.0001), normal grafts vs. Grade 1 R (AUC = 0.819, p < 0.01), Grade 2 R (AUC = 0.911, p < 0.0001), Grade 3 R (AUC = 0.996, p < 0.0001). In addition, we found changes in key enzymes and receptors of S1P pathway analysed on explanted hearts from heart failure patients. This preliminary study reveals that circulating S1P determination could be a novel approach to detect cardiac rejection, showing a robust capability for detection that improves gradually with the severity of rejection. These alterations could be relevant to better understand the involvement of calcium regulation on the pathophysiology of rejection.