This paper reports a study of the interaction between a ditopic cyclic–open-chain polyazaligand L1py and the herbicide glyphosate [N-(phosphonomethyl)glycine or PMG] by potentiometric titrations as well as calorimetry and NMR measurements. This nonsymmetrical ligand is composed of a cyclam unit (1,4,8,11-tetraazacyclotetradecane) and the linear tetraamine 1,4,8,11-tetraazaundecane linked by a 2,6-dimethylpyridine spacer. This ligand forms stable adducts with PMG. For comparison, two analogous symmetrical ditopic ligands, a bis-macrocycle L4py and a bis-tetraamine L5py, and two related anions, iminodiacetic acid (IDA) and iminobis(methylphosphonic acid) (IDP), were also investigated. The results unambiguously show that L1py selectively binds PMG, especially around neutral pH, whereas L4py and L5py exhibit a weaker affinity for this substrate. Calorimetic and 1H NMR measurements showed that the host–guest adducts are mainly stabilized by charge–charge interactions and hydrogen bonds involving the ammonium function of PMG and the nonprotonated nitrogen atom of the pyridine linker.