In higher plants, l-galactono-1,4-lactone dehydrogenase (GLDH) participates in the biosynthesis of ascorbic acid (AsA) and the assembly of mitochondrial respiratory complex I. In this work, homozygous Arabidopsis thaliana mutant (gldhRNAi3-11) plants with approximately 40 % of the GLDH activity of wild type (WT) controls were developed by RNA interference, and were found to be viable under standard laboratory conditions. Compared with WT controls, gldhRNAi3-11 plants showed about 20 % decrease in the contents of reduced AsA and total AsA. Like previously recorded for several AsA-deficient mutants (vtc1-1, vtc2-1, vtc3-1 and vtc4-1) grown under long day (LD) conditions, gldhRNAi3-11 plants exhibited an early flowering phenotype in LD environment. Interestingly, relative to WT control, vtc1-1 and vtc4-1, gldhRNAi3-11 had significantly lower leaf water loss rate. Further analysis indicated that reduced stomatal aperture size was likely responsible for the lower water loss displayed by gldhRNAi3-11 leaves. Thus, our work demonstrates that partial suppression of GLDH activity confers significant reduction in leaf water loss through decreasing stomatal aperture size in Arabidopsis. The novel phenotypes displayed by gldhRNAi3-11 plants, the new insights obtained by this work, and their implications on further study of GLDH function in higher plants are discussed.