Noxo1, the organizing element of the Nox1-dependent NADPH oxidase complex responsible for producing reactive oxygen species, has been described to be degraded by the proteasome. We mutated a D-box in Noxo1 to express a protein with limited degradation and capable of maintaining Nox1 activation. Wild-type (wt) and mutated Noxo1 (mut1) proteins were expressed in different cell lines to characterize their phenotype, functionality, and regulation. Mut1 increases ROS production through Nox1 activity affects mitochondrial organization and increases cytotoxicity in colorectal cancer cell lines. Unexpectedly the increased activity of Noxo1 is not related to a blockade of its proteasomal degradation since we were unable in our conditions to see any proteasomal degradation either for wt or mut1 Noxo1. Instead, D-box mutation mut1 leads to an increased translocation from the membrane soluble fraction to a cytoskeletal insoluble fraction compared to wt Noxo1. This mut1 localization is associated in cells with a filamentous phenotype of Noxo1, which is not observed with wt Noxo1. We found that mut1 Noxo1 associates with intermediate filaments such as keratin 18 and vimentin. In addition, Noxo1 D-Box mutation increases Nox1-dependent NADPH oxidase activity. Altogether, Nox1 D-box does not seem to be involved in Noxo1 degradation but rather related to the maintenance of the Noxo1 membrane/cytoskeleton balance.