The synthesis of a series of vinylated analogues of sphingosine-1-phosphate together with their unambiguous configurational assignment by VCD methods is reported. Among them, compound RBM10-8 can irreversibly inhibit human sphingosine-1-phosphate lyase (hS1PL) while behaving also as an enzyme substrate. These findings, together with the postulated mechanism for S1PL activity, reinforce the role of RBM10-8 as a new mechanism-based hS1PL inhibitor.