Abstract Nicotinamide adenine dinucleotide (NAD+) is a cofactor and signaling molecule that regulates critical processes from DNA repair to axon degeneration. NAD+ levels decline with mitochondrial dysfunction, in aging, and in various age- related pathologies. Moreover, studies have repeatedly shown that increased NAD+ levels are beneficial for cellular and organismal healthspan. Multiple cellular compartments contain NAD+-synthesis and NAD+-consuming signaling proteins, but little is known about the mechanisms by which NAD+ concentrations in each organelle are appropriately tuned to metabolic and signaling demands. Mitochondria harbor up to 70% of cellular NAD+, and NAD+ acts as an electron carrier in mitochondrial respiration and as a signaling molecule through the mitochondrial sirtuins. However, it is not known how the mitochondrial NAD+ pool is established and maintained or how it contributes to cellular metabolic control. Using gene co-essentiality analysis, we recently identified a mitochondrial transporter, SLC25A51, required for the uptake of NAD+ into mitochondria. We hypothesize that SLC25A51 is a major factor in cellular NAD+ compartmentalization and a critical player in metabolic homeostasis and a cell’s response to stress. A major long- term goal of our research program is to understand how cells regulate their mitochondrial NAD+ pool and NAD+ compartmentalization to control metabolism and signaling in health and disease. In the next five years, we will focus on three areas outlined in this proposal: 1) understanding the biochemical mechanism and driving forces of mitochondrial NAD+ transport by characterizing the activity of SLC25A51 in cell-free proteoliposome transport assays; 2) determining how mitochondrial NAD+ transport is regulated, by identifying the signaling pathway and transcription factors that regulate SLC25A51 expression and by elucidating how the protein is turned over in or removed from the inner mitochondrial membrane; and 3) delineating how cytosolic-mitochondrial NAD+ compartmentalization contributes to metabolic regulation in the liver using SLC25A51 loss-of-function mouse models. The results of our studies will provide important insights into the mechanisms of cellular NAD+ compartmentalization and identify new modes of metabolic regulation relevant for human disease. This research program is aligned with our laboratory’s overall goals to understand the mechanisms of metabolic compartmentalization, and specifically the role of mitochondrial transporters, in adjusting mitochondrial function to metabolic demands and cell state, and responding to stress.