l-Galactono-1, 4-lactone dehydrogenase (GalLDH; EC 1.3.2.3) is the last key enzyme in the putative l-ascorbic acid (AsA) biosynthetic pathway of higher plants. To evaluate the effect of the gene on manipulating AsA accumulation, a cDNA encoding GalLDH (RrGalLDH, Acc. No. AY643403), isolated from Rosa roxburghii fruit known to be rich in AsA, was introduced into tobacco plants by Agrobacterium-mediated transformation under CaMV 35S constitutive promoter in the present study. Southern blotting revealed the stable integration of the transgene with single copy in four independent transgenic lines, among which, L3 and L4 showed the significantly enhanced RrGalLDH transcript levels, GalLDH activities and AsA accumulations as compared to untransformed (WT) plants. So, we developed the AsA-overproducing tobacco plant by overexpressing GalLDH. As exposed to salt stress (100 mM NaCl), these AsA-overproducing transgenic lines were found to grow better with increased shoot length and fresh weight than WT. Furthermore, L3, which demonstrated the highest AsA accumulation (2.1-fold higher than WT) and expression level of RrGalLDH, showed a higher resistance to oxidative stress caused by paraquat when compared to WT. These results further justify that the overexpression of GalLDH gene confers an elevated AsA accumulation and tolerance against environmental stresses.