Co2+ binding to the nicotinamide adenine dinucleotide (NAD+) molecule in water solution was studied by electron paramagnetic resonance (EPR) and electron spin echo at low temperatures. Cobalt is coordinated by NAD+ when the metal is in excess only, but even in such conditions, the Co/NAD+ complexes coexist with Co(H2O)6 complexes. EPR spin-Hamiltonian parameters of the Co/NAD+ complex at 6 K are g z = 2.01, g x = 2.38, g y = 3.06, A z = 94 × 10−4 cm−1, A x = 33 × 10−4 cm−1 and A y = 71 × 10−4 cm−1. They indicate the low-spin Co2+ configuration with S = 1/2. Electron spin echo envelope modulation spectroscopy with Fourier transform of the modulated spin echo decay shows a strong coordination by nitrogen atoms and excludes the coordination by phosphate and/or amide groups. Thus, Co2+ ion is coordinated in pseudo-tetrahedral geometry by four nitrogen atoms of adenine rings of two NAD+ molecules.