The interplay between cardiac sarcoplasmic Ca2+ATPase and phospholamban is a key regulating factor of contraction and relaxation in the cardiac muscle. In heart failure, aberrations in the inhibition of sarcoplasmic Ca2+ATPase by phospholamban are associated with anomalies in cardiac functions. In experimental heart failure models, modulation of the interaction between these two proteins has been shown to be a potential therapeutic approach. The aim of our research was to find molecules able to interfere with the inhibitory activity of phospholamban on sarcoplasmic Ca2+ATPase. For this purpose, a portion of phospholamban was synthesized and used as target for a phage-display peptide library screening. The cyclic peptide C-Y-W-E-L-E-W-L-P-C-A was found to bind to phospholamban (1–36) with high specificity. Its functional activity was tested in Ca2+uptake assays utilizing preparations from cardiac sarcoplasmic reticulum. By synthesizing and testing a series of alanine point-mutated cyclic peptides, we identified which amino acid was important for the inhibition of the phospholamban function. The structures of active and inactive alanine-mutated cyclic peptides, and of phospholamban (1–36), were determined by NMR. This structure–activity analysis allowed building a model of phospholamban –cyclic peptide complex. Thereafter, a simple pharmacophore was defined and used for the design of small molecules. Finally, examples of such molecules were synthesized and characterized as phospholamban inhibitors.