The synthesis and characterization of the 15N‐labeled analogue of the mitochondrial calcium uptake inhibitor [Cl(NH3)4Ru(μ‐N)Ru(NH3)4Cl]3+ (Ru265) bearing [15N]NH3 ligands is reported. Using [1H,15N] HSQC NMR spectroscopy, the rate constants for the axial chlorido ligand aquation of [15N]Ru265 in pH 7.4 buffer at 25 °C were found to be k1=(3.43±0.03)×10−4 s−1 and k2=(4.03±0.09)×10−3 s−1. The reactivity of [15N]Ru265 towards biologically relevant small molecules was also assessed via this method, revealing that this complex can form coordination bonds to anionic oxygen and sulfur donors. Time‐based studies on these ligand‐binding reactions reveal this process to be slow relative to the time required for the complex to inhibit mitochondrial calcium uptake, suggesting that hydrogen‐bonding interactions, rather than the formation of coordination bonds, may play a more significant role in mediating the inhibitory properties of this complex. [ABSTRACT FROM AUTHOR]