Post-translational S-palmitoylation by the zDHHC family of palmitoyl transferases impacts the function of thousands of proteins in animals and plants. We report here that nitric oxide (NO), which is generated in organisms across phylogeny, globally diminishes S-palmitoylation. Short-term exposure to NO S-nitrosylates zDHHCs and inhibits auto-palmitoylation of the catalytic Cys within the conserved DHHC active site. Longer-term NO exposure, in particular iNOS induction in activated macrophages promotes zDHHC down-regulation via lysosomal degradation. Thus, regulation by NO of the S-palmitolome may operate across phylogeny, and dysregulated S-palmitoylation likely represents a shared concomitant of the myriad human pathophysiologies characterized by enhanced production of NO.