The multi-subunit NADPH oxidase complex plays a crucial role in host defense against microbial infection through the production of reactive oxygen species. Activation of the NADPH oxidase requires the targeting of a cytoplasmic p40-p47-p67^phox complex to the membrane bound heterodimeric p22-gp91^phox flavocytochrome. This interaction is prevented in the resting state due to an auto-inhibited conformation of p47^phox. The X-ray structure of the auto-inhibited form of p47^phox reveals that tandem SH3 domains function together to maintain the cytoplasmic complex in an inactive form. Further structural and biochemical data show that phosphorylation of p47^phox activates a molecular switch that relieves the inhibitory intramolecular interaction. This permits p47^phox to interact with the cytoplasmic tail of p22^phox and initiate formation of the active, membrane bound enzyme complex.