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In legume nodules, malate is generally considered to provide carbon for both nodule bacteroid respiration and skeletons for NH4+ assimilation. Although it has been suggested that phosphorus (P) deficiency inhibits legume nodule growth through adversely affecting carbon partitioning, the molecular mechanisms underlying malate synthesis and its roles in nodule growth remain largely unknown in soybean (Glycine max). In this study, we found that P deficiency led to a significant decrease of soybean nodule size (fresh weight of individual soybean nodule), accompanying with increased malate concentration. Meanwhile, 16 malate dehydrogenase (GmMDH) members were identified in the soybean genome, and their expression patterns were investigated in soybean nodules in responses to P deficiency. Among them, GmMDH12 was up-regulated by phosphate (Pi) starvation. Enzymatic analysis of GmMDH12 revealed its functions in catalyzing malate synthesis through reduction of oxaloacetate to form malate in vitro. Furthermore, overexpression of GmMDH12 resulted in a significant decrease in nodule size, but a significant increase in concentrations of malate and several carbohydrates in transgenic soybean nodules. Taken together, these results suggest that P deficiency inhibits soybean nodule size due to enhancement of malate synthesis, which is regulated by GmMDH12 transcripts.