Mn 2+ complexes of 2,4‐pyridyl‐disubstituted bispidine ligands have emerged as more biocompatible alternatives to Gd 3+ ‐based MRI probes. They display relaxivities comparable to that of commercial contrast agents and high kinetic inertness, unprecedented for Mn 2+ complexes. The chemical structure, in particular the substituents on the two macrocyclic nitrogens N3 and N7, are decisive for the conformation of the Mn 2+ complexes, and this will in turn determine their thermodynamic, kinetic and relaxation properties. We describe the synthesis of four ligands with acetate substituents in positions N3, N7 or both. We evidence that the bispidine conformation is dependent on N3 substitution, with direct impact on the thermodynamic stability, kinetic inertness, hydration state and relaxivity of the Mn 2+ complexes. These results unambiguously show that (i) solely a chair‐chair conformation allows for favorable inertness and relaxivity, and (ii) in this family such chair‐chair conformation is accessible only for ligands without N3‐appended carboxylates.