Nicotinamide (NAM) is the main precursor of nicotinamide adenine dinucleotide (NADþ), a coenzyme essential for DNA repair, glycolysis, and oxidative phosphorylation. NAM has anti-aging activity on human skin, but the underlying mechanisms of action are unclear. Using 3-dimensional organotypic skin models, we show that NAM inhibits differentiation of the upper epidermal layers and maintains proliferation in the basal layer. In 2-dimensional culture, NAM reduces the expression of early and late epidermal differentiation markers and increases the proliferative capacity of human primary keratinocytes. This effect is characterized by elevated clonogenicity and an increased proportion of human primary keratinocyte stem cell (holoclones) compared to controls. By contrast, preventing the conversion of NAM to NADþ using FK866 leads to premature human primary keratinocyte differentiation and senescence, together with a dramatic drop in glycolysis and cellular adenosine triphosphate levels while oxidative phosphorylation is moderately affected. All these effects are rescued by addition of NAM, known to compete with FK866, which suggests that conversion to NADþ is part of the mechanistic response. These data provide insights into the control of differentiation, proliferation, and senescence by NAM and NADþ in skin. They may lead to new therapeutic advances for skin conditions characterized by dysregulated epidermal homeostasis and premature skin aging, such as photoaging.