Phagocyte-like NADPH oxidase (Nox2) has been shown to play regulatory roles in the metabolic dysfunction of the islet β-cell under the duress of glucolipotoxic conditions and exposure to proinflammatory cytokines. However, the precise mechanisms underlying Nox2 activation by these stimuli remain less understood. To this end, we report a time-dependent phosphorylation of p47phox, a cytosolic subunit of Nox2, by cytomix (IL-1β+TNFα+IFNγ) in insulin-secreting INS-1 832/13 cells. Furthermore, cytomix induced the expression of gp91phox, a membrane component of Nox2. 2-Bromopalmitate (2-BP), a known inhibitor of protein palmitoylation, markedly attenuated cytokine-induced, Nox2-mediated reactive oxygen species (ROS) generation and inducible nitric oxide synthase (iNOS)-mediated nitric oxide (NO) generation. However, 2-BP failed to exert any significant effects on cytomix-induced CHOP expression, a marker for endoplasmic reticulum stress. Together, our findings identify palmitoyltransferase as a target for inhibition of cytomix-induced oxidative (ROS generation) and nitrosative (NO generation) stress in the pancreatic β-cell.