Nicotinamide adenine dinucleotide (NAD+) spans diverse roles in biology, serving as both an important redox cofactor in metabolism and a substrate for signaling enzymes that regulate protein post-translational modifications (PTMs). Although the interactions between these different roles of NAD+ (and its reduced form NADH) have been considered, little attention has been paid to the role of compartmentation in these processes. Specifically, the role of NAD+ in metabolism is compartment specific (e.g., mitochondrial vs. cytosolic), affording a very different redox landscape for PTM-modulating enzymes such as sirtuins and poly(ADP-ribose) polymerases in different cell compartments. In addition, the orders of magnitude differences in expression levels between NAD+-dependent enzymes are often not considered when assuming the effects of bulk changes in NAD+ levels on their relative activities. In this review, we discuss the metabolic, nonmetabolic, redox, and enzyme substrate roles of cellular NAD+, and the recent discoveries regarding the interplay between these roles in different cell compartments.