Ascorbate (AsA) is an important antioxidant that can scavenge reactive oxygen species to protect plant cells against oxidative stress. GDP-L-galactose phosphorylase (GGP) is a key enzyme in the AsA biosynthetic pathway. To investigate the functions of GGP in AsA synthesis and oxidative stress tolerance in tomato, antisense lines with reduced expression of GDP-L-galactose phosphorylase gene (SlGGP) were obtained. Photobleaching after treatment of leaf disks with methyl viologen was more severe in transgenic lines compared to wild type (WT) plants. Moreover, compared with WT plants, transgenic plants showed higher content of hydrogen peroxide, superoxide anion, malondialdehyde as well as ion leakage, but lower content of AsA and chlorophylls, ascorbate peroxidase activity, net photosynthetic rate, and maximal photochemical efficiency of photosystem II. Results of real-time quantitative polymerase chain reaction showed that suppression of SlGGP gene in transgenic plants reduced their oxidative stress tolerance.