NOX (NADPH oxidases) are a family of NADPH-dependent transmembrane enzymes that synthesize superoxide and other reactive oxygen species. There are seven isoforms (NOX1–5 and DUOX1–2) which derive from a common ancestral NOX. NOX enzymes are distinguished by different modes of activation, the types of ROS that are produced, the cell types where they are expressed, and distinct functional roles. NOX5 was one of the earliest eukaryotic Nox enzymes to evolve and ironically the last isoform to be discovered in humans. In the time since its discovery, our knowledge of the regulation of NOX5 has expanded tremendously, and we now have a more comprehensive understanding of the molecular mechanisms underlying NOX5-dependent ROS production. In contrast, the cell types where NOX5 is robustly expressed and its functional significance in health and disease remain an underdeveloped area. The goal of this chapter is to provide an up-to-date overview of the mechanisms regulating NOX5 function and its importance in human physiology and pathophysiology.