Nicotinamide adenine dinucleotide (NAD) is an essential co-enzyme mediating various enzymatic reactions. Mitochondrial NAD particularly occupies a considerable amount of total NAD in cells, and serves as a co-enzyme in tricarboxylic acid cycle (TCA cycle), β-oxidation, and oxidative phosphorylation. Despite the importance of mitochondrial NAD, its synthesis pathway remains unknown. It has been proposed that NAD synthesis enzyme, Nmnat3, was localized in mitochondria, but its physiological relevance to the metabolism in mitochondria was not fully elucidated. Previously, we have reported that murine Nmnat3 protein was strongly expressed in the cytoplasm of mature erythrocytes, in which mitochondria were absent, and Nmnat3-deficient mice (Nmnat3-KO mice) exhibited splenomegaly and hemolytic anemia due to reduced NAD levels in mature erythrocytes. These results challenged the role of Nmnat3 in mitochondrial NAD synthesis. In this study, we demonstrated that mitochondrial NAD levels in various tissues, except for red blood cells, were unchanged in Nmnat3-KO mice. We also analyzed the metabolites in glycolysis and TCA cycle and found that there were no differences between Nmnat3-KO and WT mice. In addition, the aged Nmnat3-KO mice had comparable NAD levels to that observed in WT mice. Our results indicated that Nmnat3 is dispensable in the maintenance of mitochondrial NAD levels, and that other NAD regulatory pathways may exist in mitochondria.