To determine the rates of cellular NAD⁺ synthesis and breakdown, incorporation of stable isotope-labeled precursors into NAD⁺ should be quantified. Although with ²H (D)-labeled precursors [2,4,5,6-D₄]nicotinamide ([D₄]Nam) and [2,4,5,6-D₄]nicotinic acid ([D₄]NA), [D₃]NAD⁺ is formed in human cells, why only three of four D atoms from [D₄]Nam and [D₄]NA are present in NAD⁺ remains unknown. Using a liquid chromatography-tandem mass spectrometry, we tested the involvement of D/¹H (H) exchange at the redox site of NAD⁺/NADH (C-4 carbon of the pyridine ring) by oxidoreductases exhibiting opposite stereospecificity for the coenzymes in the 1-Da mass decrease in the cellular NAD⁺ formation. In all cells examined, [Nam-D₃]NAD⁺, but not [Nam-D₄]NAD⁺, was obtained after the incubation with the D₄-labeled precursors, whereas [Nam-D₄]NAD⁺, but not [Nam-D₃]NAD⁺, was synthesized from the same precursors with purified recombinant NAD⁺ biosynthetic enzymes. [D₄]Nam group of [Nam-D₄]NAD⁺ was converted to [D₃]Nam group via [D₄]NADH by in vitro sequential reduction and oxidation with oxidoreductases exhibiting opposite stereospecificity for the coenzymes. Furthermore, using [2,5,6-D₃]Nam, which has H instead of D at the C-4 carbon, as a precursor of NAD⁺ in the cells, the 1-Da mass decrease in the nucleotide was not observed. Based on these observations, we conclude that following the synthesis of [Nam-2,4,5,6-D₄]NAD⁺, cellular redox reactions of NAD⁺/NADH convert [Nam-2,4,5,6-D₄]NAD⁺ to [Nam-2,5,6-D₃]NAD⁺. Quantification of [Nam-2,5,6-D]NAD⁺ and [2,5,6-D₃]Nam would successfully determine the rate of the NAD⁺ turnover and provide clues to understand regulatory mechanisms of cellular NAD⁺ concentrations.