Non-phosphorylating glyceraldehyde- 3-phosphate dehydrogenase (NP-GAPDH) is a conserved cytosolic protein found in higher plants. In photosynthetic cells, the enzyme is involved in a shuttle transfer mechanism to export NADPH from the chloroplast to the cytosol. To investigate the role of this enzyme in plant tissues, we characterized a mutant from Arabidopsis thaliana having an insertion at the NP-GAPDH gene locus. The homozygous mutant was determined to be null respect to NP-GAPDH, as it exhibited undetectable levels of both transcription of NP-GAPDH mRNA, protein expression and enzyme activity. Transcriptome analysis demonstrated that the insertion mutant plant shows altered expression of several enzymes involved in carbohydrate metabolism. Significantly, cytosolic phosphorylating (NAD-dependent) glyceraldehyde-3-phosphate dehydrogenase mRNA levels are induced in the mutant, which correlates with an increase in enzyme activity. mRNA levels and enzymatic activity of glucose-6-phosphate dehydrogenase were also elevated, correlating with an increase in NADPH concentration. Moreover, increased ROS levels were measured in the mutant plants. Down-regulation of several glycolytic and photosynthetic genes suggests that NP-GAPDH is important for the efficiency of both metabolic processes. The results presented demonstrate that NP-GAPDH has a relevant role in plant growth and development.