The NADPH oxidase is the sole enzymatic complex that produces, in a controlled way, superoxide anions. In phagocytes, it is constituted by the assembly of four cytosolic (p67phox, p47phox, p40phox and Rac) and two membrane (p22phox and Nox2) proteins. In response to pro‐inflammatory mediators, the NADPH oxidase is activated. In cells, arachidonic acid (cis‐AA), released by activated phospholipase A2, also plays a role in activation of the NADPH oxidase complex, but the mechanism of action of cis‐AA is still a matter for debate. In cell‐free systems, cis‐AA is commonly used for activation. We have shown previously that trans‐AA isomers were unable to activate the NADPH oxidase complex. Here, we aim to evaluate the structural changes in p47phox and p67phox induced by AA. The structural impact of both AA isomers on both cytosolic proteins was investigated by the accessibility of the thiol group and by circular dichroism in the far‐UV for global folds. cis‐AA induces secondary structure changes of p47phox and p67phox, while the trans isomer does not, suggesting that the changes observed are of importance for the activation process of these proteins. While five of the nine thiol groups in p67phox and all of them in p47phox have low access to the solvent when proteins are alone in solution, all of them become fully accessible when proteins are together. In conclusion, the secondary structures of p47phox and p67phox are both dependent on the presence of the partner protein in solution and on the presence of the activator molecule cis‐AA. Structure of resting/activated p47phox and p67phox: when not activated, the interactions between both proteins increase their flexibility but not their secondary structures. Upon arachidonic acid activation, their secondary structures individually are modified but not the global architecture of heterodimer. Protein activation depends not only on arachidonic acid but also on the presence of the cytosolic partner protein.