A new diethylenetriamine-derived macrocycle bearing 2-methylpyridyl arms and containing m-xylyl spacers, L, was prepared, and its dinuclear copper(II) and zinc(II) complexes were used as receptors for the recognition in aqueous solution of a phosphorylated peptide derived from a sequence of the STAT3 protein. A detailed study of the acid-base behavior of L and of its complexation properties as well as of the association of the phosphorylated peptide to the receptor was carried out by potentiometry in aqueous solution at 298.2 K and I = 0.10 M in KNO3. The data revealed that the receptor forms stable associations with several protonated forms of the substrate, with constant values ranging from 3.32 to 4.25 log units. The affinity of the receptor for the phosphorylated substrate studied is higher at a pH value where the receptor is mainly in the [Cu2L](4+) form and the pY residue of the substrate is in the dianionic form (pH 6.55). These results, also supported by (31)P NMR studies, showed that the phosphopeptide is bound through the phosphoryl group in a bridging mode. Additionally, the receptor inhibited binding between active (phosphorylated) STAT3 and its target DNA sequence in a dose-dependent manner (IC50 63 ± 3.4 μM) in human nuclear extracts in vitro. Treatment of whole cells with the inhibitor revealed that it is bioactive in living cells and has oncostatic properties that could be interesting for the fight against cancer and other pathologies involving the STAT3 protein.