Sphingosine-1-phosphate (S1P) is a bioactive lipid with key functions in the immune, inflammatory, and cardiovascular systems. S1P exerts its action through the interaction with a family of five known G protein-coupled receptors, named S1P1–5. Among them, S1P3 has been implicated in the pathological processes of a number of diseases, including sepsis and cancer. KRX-725 (compound 1) is a pepducin that mimics the effects of S1P by triggering specifically S1P3. Here, aiming to identify novel S1P3 antagonists, we carried out an alanine scanning analysis to address the contribution of the side chains of each amino acid residue to the peptide function. Then, deleted peptides from both the C- and N-terminus were prepared in order to determine the minimal sequence for activity and to identify the structural requirements for agonistic and, possibly, antagonistic behaviors. The pharmacological results of the Ala-scan derived compounds (2–10) suggested a high tolerance of the pepducin 1 to amino acid substitutions. Importantly, the deleted peptide 16 has the ability to inhibit, in a dose-dependent manner, both pepducin 1-induced vasorelaxation and fibroblast proliferation. Finally, a computational analysis was performed on the prepared compounds, showing that the supposed antagonists 16 and 17 appeared to be aligned with each other but not with the others. These results suggested a correlation between specific conformations and activities. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.