The flavoprotein l-aspartate oxidase (LASPO) is the first enzyme of the de novo biosynthetic pathway of NAD+ in plants. Although LASPO is considered pivotal to maintain NAD+ homeostasis, it has not been hitherto characterized in plants. Here, the cDNA encoding the LASPO from the model plant Arabidopsis thaliana (AtLASPO, At5g14760) has been cloned and expressed in Escherichia coli for subsequent enzyme characterization. The purified AtLASPO enzyme displayed a Km of 0.79 mM for l-aspartate and a kcat of 0.25 s−1. We could further detect an l-aspartate: fumarate oxidoreductase activity of the recombinant plant enzyme. In addition, results indicated that NADP+ but not NAD+, and even more strongly NADH, inhibited AtLASPO at physiological concentrations by competing with the flavin for binding to the apoprotein. LASPO optimal pH and temperature, as well as plastidial pyridine nucleotide concentrations may contribute to an increased NAD+ production in planta. Moreover, in Arabidopsis thaliana AtLASPO gene expression exhibited a clear correlation between LASPO activity and NAD+ levels, thus demonstrating that plant LASPO catalyzes a key metabolic step of NAD+ synthesis.