Abstract: Nicotinamide adenine dinucleotide (NAD+) is an essential cofactor involved in various cellular biochemical reactions. To date, the signaling pathways that regulate NAD+ metabolism remain unclear due to the dynamic nature and complexity of the NAD+ metabolic pathways and the difficulty of determining the levels of the interconvertible pyridine nucleotides. Nicotinamide riboside (NmR) is a key pyridine metabolite that is excreted and re-assimilated by yeast and plays important roles in the maintenance of NAD+ pool. In this study, we establish a NmR-specific reporter system and use it to identify yeast mutants with altered NmR/NAD+ metabolism. We show that the phosphate responsive signaling (PHO) pathway contributes to control NAD+ metabolism. Yeast strains with activated PHO pathway show increases in both the release rate and internal concentration of NmR. We further identify Pho8, a PHO-regulated vacuolar phosphatase, as a major NmR production factor. We also demonstrate that Fun26, a homolog of human ENT (equilibrative nucleoside transporter), localizes to the vacuolar membrane and establishes the size of the vacuolar and cytosolic NmR pools. In addition, the PHO pathway responds to depletion of cellular NaMN (nicotinic acid mononucleotide) and mediates NMN (nicotinamide mononucleotide) catabolism, thereby contributing to both NmR salvage and phosphate acquisition. Therefore, NaMN is a putative molecular link connecting the PHO signaling and NAD+ metabolic pathways. Our findings may contribute to the understanding of the molecular basis and regulation of NAD+ metabolism in higher eukaryotes.