A mitochondrial glycerol-3-phosphate (G-3-P) shuttle that channels cytosolic reducing equivalent to mitochondria for respiration through oxidoreduction of G-3-P has been extensively studied in yeast and animal systems. Here, we report evidence for the operation of such a shuttle in Arabidopsis thaliana. We studied Arabidopsis mutants defective in a cytosolic G-3-P dehydrogenase, GPDHc1, which, based on models described for other systems, functions as the cytosolic component of a G-3-P shuttle. We found that the gpdhc1 T-DNA insertional mutants exhibited increased NADH/NAD+ ratios compared with wild-type plants under standard growth conditions, as well as impaired adjustment of NADH/NAD+ ratios under stress simulated by abscisic acid treatment. The altered redox state of the NAD(H) pool was correlated with shifts in the profiles of metabolites concerning intracellular redox exchange. The impairment in maintaining cellular redox homeostasis was manifest by a higher steady state level of reactive oxygen species under standard growth conditions and by a significantly augmented hydrogen peroxide production under stress. Loss of GPDHc1 affected mitochondrial respiration, particularly through a diminished capacity of the alternative oxidase respiration pathway. We propose a model that outlines potential involvements of a mitochondrial G-3-P shuttle in plant cells for redox homeostasis.