Rotenone, a widely used pesticide, reproduces parkinsonism in rodents and associates with increased risk for Parkinson disease. We previously reported that rotenone increased superoxide production by stimulating the microglial phagocyte NADPH oxidase (PHOX). This study identified a novel mechanism by which rotenone activates PHOX. Ligand-binding assay revealed that rotenone directly bound to membrane gp91 phox, the catalytic subunit of PHOX; such binding was inhibited by diphenyleneiodonium, a PHOX inhibitor with a binding site on gp91 phox. Functional studies showed that both membrane and cytosolic subunits were required for rotenone-induced superoxide production in cell-free systems, intact phagocytes, and COS7 cells transfected with membrane subunits (gp91 phox/p22 phox) and cytosolic subunits (p67 phox and p47 phox). Rotenone-elicited extracellular superoxide release in p47 phox-deficient macrophages suggested that rotenone enabled activation of PHOX through a p47 phox-independent mechanism. Increased membrane translocation of p67 phox, elevated binding of p67 phox to rotenone-treated membrane fractions, and coimmunoprecipitation of p67 phox and gp91 phox in rotenone-treated wild-type and p47 phox-deficient macrophages indicated that p67 phox played a critical role in rotenone-induced PHOX activation via its direct interaction with gp91 phox. Rac1, a Rho-like small GTPase, enhanced p67 phox–gp91 phox interaction; Rac1 inhibition decreased rotenone-elicited superoxide release. In conclusion, rotenone directly interacted with gp91 phox; such an interaction triggered membrane translocation of p67 phox, leading to PHOX activation and superoxide production.