Reduced L-ascorbic acid can be oxidized by ascorbate peroxidase (APX) in ascorbate metabolism pathway. In present study, a mitochondrial APX (mitAPX) was cloned and suppressed 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. Decreased mitAPX expression in homozygous transgenic lines was detected by semi-quantitative reverse transcriptase-polymerase chain reaction, accompanied by decreasing cytosolic APX activity by 0.4- to 0.8-fold of the untransformed control. The mitochondrial APX enzyme activity in RNAi lines decreased to 29-45% of the wild type plants. The down-regulated mitAPX expression and APX enzyme activity lead to 1.4- to 2.2-fold increase in ascorbic acid content in tomato fruits compared with wild-type control. The APX expression and APX enzyme activity were found to be correlated with increase in the amount of ascorbic acid in tomato fruits. This report demonstrates the modulation of ascorbic acid metabolic pathway by RNA interference method, and provides a feasible approach to improve ascorbic acid content in horticultural plants by suppressing genes related to ascorbic acid metabolism. (C) 2011 Elsevier B.V. All rights reserved.