The physiological role of mitochondrial uncoupling protein-3 (UCP3) is debated. UCP3 seem to be involved in a mild uncoupling of oxidative phoshorylation by mediating the leak of protons across the mitochondrial inner membrane (MIM). However, an increased expression of UCP3 has not always been associated with mitochondrial uncoupling, thus suggesting that uncoupling is not the primary function of UCP3, but rather a consequence of it. We tested the hypothesis that UCP3 is involved in the export of lipid hydroperoxides (LOOH) across the MIM and that this process is also associated to LOOH-dependent UCP3-mediated mitochondrial uncoupling. To this aim we performed a study on skeletal muscle mitochondria from UCP3-/- and UCP3+/+ mice. In experimental condition in which high level of LOOH are formed at the matrix side of the MIM, UCP3+/+ mitochondria released a significant more LOOH than UCP3-/-. These differences were abolished when UCP3 was inhibited by GDP (that was ineffective in UCP3-/- mitochondria) and when the formation of LOOH at the matrix side of the MIM was prevented. Tha data demonstrate that UCP3 plays a role in the extrusion of LOOH across the MIM. When high level of LOOH are formed at the matrix side of the MIM, UCP3+/+ mitochondria showed an enhanced proton leak compared to UCP3-/-ones. These differences were abolished by GDP or by preventing LOOH formation. We conclude that LOOH formation is a key event in the activation of UCP3-mediated proton leak.