Plant mitochondrial uncoupling proteins (pUCPs) play important roles in generation of metabolic thermogenesis, response to stress situation, and regulation of energy metabolism. Although the signaling pathways for the pUCPs-regulated plant energy metabolism and thermogenesis are well studied, the role of pUCPs in the regulation of plant stress tolerance has not been fully substantiated. Here we showed that mitochondrial uncoupling protein was required for effective antioxidant enzymes activities, chlorophyll fluorescence and redox poise in tomato under oxidative stress using virusinduced gene silencing approach. Silencing of LeUCP gene reduced maximal quantum yield of PSII (Fv/Fm) and photochemical quenching coefficient (qP), as well as mitigated activation of antioxidant enzymes and related genes expression. The content of reduced ascorbate and reduced glutathione, redox ratio of ascorbate and L-galactono-1,4-lactone dehydrogenase (GalLDH; EC 1.3.2.3) activity were all decreased in the leaves of LeUCP gene-silenced plant. However, malondialdehyde content was increased under methylviologen (MV) stress. ROS accumulation was increased significantly following MV and heat stress treatments. Meanwhile, LeUCP gene silencing aggravated accumulation of H2O2 and O 2 ·− in leaves. Taken together, these results strongly suggest that LeUCP gene plays critical role in maintaining the redox homeostasis and balance in antioxidant enzyme system under oxidative stress.