Several uncoupling protein isoforms were reported to be activated by oxidative stress, particularly by 4‑hydroxy-2-nonenal (HNE), a product of lipid peroxidation generated by superoxide. The recently identified gene of Arabidopsis thaliana uncoupling protein 5 (AtPUMP5) was strongly induced by stressful conditions. To better understand the role of AtPUMP5 in response of plant to stress situations, its gene was cloned and expressed in Escherichia coli BL21(DE3) pLysS strain. The recombinant protein was purified from inclusion bodies, reconstituted into proteoliposomes, and the HNE effect on its activity was investigated. The analysis of proton fluxes at pH 7.2 measured as fluorescence changes of SPQ revealed that linoleic acid (LA) induced H⁺ fluxes with an apparent K_m of 45 µM. HNE itself was not able to induce AtPUMP5-mediated H⁺ flux. The presence of HNE did not change the affinity of AtPUMP for LA, but HNE increased the rate of proton fluxes in a dose dependent manner at all LA concentrations tested. Comparing to other biochemically characterized PUMPs, AtPUMP5 presents lower sensitivity to purine nucleotides (K_i,ATP = 15.0 mM and  K_i,GDP = 10.0 mM). Inhibition by ATP was not modified by the presence of 93 µM HNE (K_i,ATP = 14.8 mM). In conclusion, HNE appears to stimulate AtPUMP5-mediated H⁺ flux induced by LA. The higher PUMP activity can control more efficiently the reactive species production by respiratory chain observed under stress conditions. Thus, AtPUMP5 may play a physiological role in the oxidative stress response as a negative feedback regulator of superoxide overproduction.