The heterolytic and homolytic C4-H bond dissociation energies of NADH and its radical cation (NADH*+) in aqueous solution were estimated according to the reaction of NADH with N,N,N',N'-tetramethyl-p-phenylenediamine radical cation perchlorate (TMPA*+) in aqueous solution. The results show that the values of the heterolytic and homolytic C4-H bond dissociation energies of NADH in aqueous solution are 53.6 and 79.3 kcal/mol, respectively; the values of the heterolytic and homolytic C4-H bond dissociation energies of NADH*+*+ in aqueous solution are 5.1 and 36.3 kcal/mol, respectively, which, to our knowledge, is first reported. This energetic information disclosed in the present work should be believed to furnish hints to the understanding of the mechanisms for the redox interconversions of coenzyme couple NADH/NAD+ in vivo.