We have previously shown that avian uncoupling protein (avUCP) is down-regulated on exposure to acute heat stress stimulating mitochondrial reactive oxygen species (ROS) production and oxidative damage. In this study, we investigated whether up-regulation of avUCP could attenuate oxidative damage caused by acute heat stress. Broiler chickens (Gallus gallus) were fed either a control diet or an olive oil-supplemented diet (6.7%), which has been shown to increase the expression of UCP3 in mammals, for 8 days and then exposed either to heat stress (34°C, 12 h) or kept at a thermoneutral temperature (25°C). Skeletal muscle mitochondrial ROS (measured as H₂O₂) production, avUCP expression, oxidative damage, mitochondrial membrane potential and oxygen consumption were studied. We confirmed that heat stress increased mitochondrial ROS production and malondialdehyde levels, and decreased amount of avUCP. As expected, feeding birds an olive oil-supplemented diet increased the expression of avUCP in skeletal muscle mitochondria, and decreased ROS production and oxidative damage. Studies on mitochondrial function showed that heat stress increased membrane potential in state 4, which were reversed by feeding birds with olive oil-supplemented diet, even though no differences in basal proton leak were observed between control and heat-stressed groups. These results show that under heat stress, mitochondrial ROS production and olive oil-induced reduction of ROS production may occur due to changes in respiratory chain activity as well as avUCP expression in skeletal muscle mitochondria.