Nicotinamide Adenine Dinucleotide (NAD⁺) is known mainly as coenzyme of redox reactions for energy transduction and is consumed as substrate in regulatory reactions removing nicotinamide and producing ADP-ribose. Several families of ADP-ribose synthesizing enzymes use NAD⁺ as substrate and control processes like DNA repair, replication and transcription, chromatin structure, the activity of G-proteins and others. Since NAD⁺-dependent reactions involve degradation of the dinucleotide, a constant supply of the pyridinic substrate is required for its homeostasis. NAD⁺-dependent signaling reactions include protein deacetylation by sirtuins, intracellular calcium signaling and mono-/poly-ADP-ribosylation. In the context of all NAD⁺-dependent reactions leading to ADP-ribose synthesis, this review focuses mainly on both the central role played by sirtuins and poly-ADPribose polymerases as cellular NAD⁺ consumers and their crosstalk in signaling pathways.