NADPH oxidases (NOXs) are specialized enzymes that transfer electrons from intracellular NADPH to extracellular oxygen, resulting in the production of superoxide anions or hydrogen peroxide. NOX2, primarily expressed in phagocytes, plays a critical role in various physiological processes and is associated with several human diseases. The activation of NOX2 is a tightly regulated process involving membrane translocation and the binding of several cytosolic factors, including p47phox, p67phox, p40phox, and Rac1. Recent advances in the structural determination of NOX2 in both its resting and activated states have elucidated the fundamental mechanisms underlying NOX2 activation. The concerted binding of cytosolic factors to NOX2 not only facilitates the docking of the dehydrogenase domain to the transmembrane domain but also reduces the distance between NADPH and FAD, thereby enhancing electron transfer efficiency. Additional structural studies of other NOX proteins shed light on their diverse activation mechanism.