In plants, glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the interconversion of glycerol-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) coupled to the reduction/oxidation of the nicotinamide adenine dinucleotide (NADH) pool, and plays a central role in glycerolipid metabolism and stress response. Previous studies have focused mainly on the NAD+-dependent GPDH isoforms, neglecting the role of flavin adenine dinucleotide (FAD)-dependent GPDHs. We isolated and characterized three mitochondrial-targeted FAD-GPDHs in soybean, of which one isoform (GmGPDH12) showed a significant transcriptional response to NaCl and mannitol treatments, suggesting the existence of a major FAD-GPDH isoform acting in soybean responses to salt and osmotic stress. An enzyme kinetic assay showed that the purified GmGPDH12 protein possessed the capacity to oxidize G3P to DHAP in the presence of FAD. Overexpression and RNA interference of GmGPDH12 in soybean hairy roots resulted in elevated tolerance and sensitivity to salt and osmotic stress, respectively. G3P contents were significantly lower in GmGPDH12-overexpressing hair roots and higher in knockdown hair roots, indicating that GmGPDH12 was essential for G3P catabolism. A significant perturbation in redox status of NADH, ascorbic acid (ASA) and glutathione (GSH) pools was observed in GmGPDH12-knockdown plants under stress conditions. The impaired redox balance was manifested by higher reactive oxygen species generation and consequent cell damage or death; however, overexpressing plants showed the opposite results for these traits. GmGPDH12 overexpression contributed to maintaining constant respiration rates under salt or osmotic stress by regulating mRNA levels of key mitochondrial respiratory enzymes. This study provides new evidence for the roles of mitochondria-localized GmGPDH12 in conferring resistance to salt or osmotic stress by maintaining cellular redox homeostasis, protecting cells and respiration from oxidative injury.