Nicotinamide phosphoribosyltransferase (NAMPT), the key NAD+ biosynthetic enzyme, has two different forms, intra- and extracellular (iNAMPT and eNAMPT), in mammals. However, the significance of eNAMPT secretion remains unclear. Here we demonstrate that deacetylation of iNAMPT by the mammalian NAD+-dependent deacetylase SIRT1 predisposes the protein to secretion in adipocytes. NAMPT mutants reveal that SIRT1 deacetylates lysine 53 (K53) and enhances eNAMPT activity and secretion. Adipose tissue-specific Nampt knockout and knockin (ANKO and ANKI) mice show reciprocal changes in circulating eNAMPT, affecting hypothalamic NAD+/SIRT1 signaling and physical activity accordingly. The defect in physical activity observed in ANKO mice is ameliorated by nicotinamide mononucleotide (NMN). Furthermore, administration of a NAMPT-neutralizing antibody decreases hypothalamic NAD+ production, and treating ex vivo hypothalamic explants with purified eNAMPT enhances NAD+, SIRT1 activity, and neural activation. Thus, our findings indicate a critical role of adipose tissue as a modulator for the regulation of NAD+ biosynthesis at a systemic level. Display omitted •SIRT1 deacetylates iNAMPT and enhances eNAMPT activity and secretion in adipocytes•eNAMPT from adipose tissue affects hypothalamic NAD+/SIRT1 and physical activity•A NAMPT-neutralizing antibody suppresses eNAMPT and hypothalamic NAD+ in vivo•eNAMPT enhances NAD+/SIRT1 and neural activity in ex vivo hypothalamic explants Yoon et al. shed light on the two forms of the NAD+ biosynthetic enzyme, intra- and extracellular NAMPT. SIRT1-mediated deacetylation of iNAMPT promotes its extracellular secretion from adipocytes. eNAMPT, in turn, affects NAD+/SIRT1 signaling in the hypothalamus, with effects on physical activity, implicating adipose tissue as a systemic NAD+ modulator.