The conceptual evolution of nicotinamide adenine dinucleotide (NAD(+)) from being seen as a simple metabolic cofactor to a pivotal cosubstrate for proteins regulating metabolism and longevity, including the sirtuin family of protein deacylases, has led to a new wave of scientific interest in NAD(+). NAD(+) levels decline during ageing, and alterations in NAD(+) homeostasis can be found in virtually all age-related diseases, including neurodegeneration, diabetes and cancer. In preclinical settings, various strategies to increase NAD(+) levels have shown beneficial effects, thus starting a competitive race to discover marketable NAD(+) boosters to improve healthspan and lifespan. Here, we review the basics of NAD(+) biochemistry and metabolism, and its roles in health and disease, and we discuss current challenges and the future translational potential of NAD(+) research.