Activation of the multicomponent enzyme NADPH oxidase requires the interaction between the tandem SH3 domain of the cytosolic subunit p47(phox) and the cytoplasmic tail of membrane-bound p22(phox). In the resting state, p47(phox) exists in an autoinhibited conformation stabilized by intramolecular contacts between the SH3 domains and an adjacent polybasic region. Phosphorylation of three serine residues, Ser(303), Ser(304), and Ser(328) within this polybasic region has been shown to be sufficient for the disruption of the intramolecular interactions thereby inducing an active state of p47(phox). This active conformation is accessible to the cytoplasmic tail of p22(phox) and initiates the formation of the membrane-bound functional enzyme complex. Molecular dynamics simulations reveal insights in the mechanism of activation of the autoinhibited form of p47(phox) by in silico phosphorylation, of the three serine residues, Ser(303), Ser(304), and Ser(328). The simulations highlight the major collective coordinates generating the opening and the closing of the two SH3 domains and the residues that cause the unmasking of the p22(phox) binding site.