Nicotinamide adenine dinucleotide (NAD+) and mitochondrial number play a vital role in cellular processes that govern human health and disease. Depletion in NAD+ levels and mitochondrial number occur with aging and metabolic diseases. Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in NAD+ biosynthesis. Thus, boosting NAD+ level and mitochondrial number with their activator(s) is an attractive approach for countering the effects of metabolic stress. This study aimed to establish IRW (Ile-Arg-Trp), a small tripeptide derived from ovotransferrin, as an activator of NAMPT and mitochondrial biogenesis. Treatment of mammalian cells with IRW increased intracellular NAMPT protein levels (p<0.05) and boosted mitochondrial biogenesis (p<0.01). A significantly increased level of circulating NAD+ and mitochondria was also observed following IRW treatment in obese mice (p<0.0001). Dosing of Drosophila melanogaster with IRW elevated both D-NAAM (fly NAMPT), NAD+, and mitochondrial content in vivo (p<0.05). The CRISPR-Cas based KO study showed the ability of IRW to activate mitochondrial biogenesis was dependent on FAM120B. Overall, these data indicate that IRW is a novel small peptide activator of mitochondrial and metabolic activity.