Reduced ascorbic acid (AsA) can be converted into oxidized state by ascorbate oxidase (AO). In present study, AO gene was down-regulated by RNA interference in tomato via Agrobacterium-mediated transformation. The existence of the RNA interference T-DNA in transformed plants and their progenies were confirmed by polymerase chain reaction amplification and Southern blotting. AO expression levels in transgenic lines were monitored by semi-quantitative reverse transcriptase-polymerase chain reaction. The decreased AO enzyme activity and significantly improved AsA content in tomato fruit were observed to be correlated with AO gene suppression. Transgenic plants with AO suppression and AsA improvement exhibited higher photosynthesis capacity than untransformed control under drought stress. This report demonstrates the AsA oxidation pathway by RNA interference, and provides a feasible approach to improve AsA content in horticultural plants by regulating genes related to AsA metabolism.