During P deficiency, the increased activity of malate dehydrogenase (MDH, EC 1.1.1.37) can lead to malate accumulation. Cytosolic- and nodule-enhanced MDH (cMDH and neMDH, respectively) are known isoforms, which contribute to MDH activity in root nodules. The aim of this study was to investigate the role of the cMDH isoforms in nodule malate supply under P deficiency. Nodulated lupins (Lupinus angustifolius var. Tanjil) were hydroponically grown at adequate P (+P) or low P (−P). Total P concentration in nodules decreased under P deficiency, which coincided with an increase in total MDH activity. A consequence of higher MDH activity was the enhanced accumulation of malate derived from dark CO2 fixation via PEPC and not from pyruvate. Although no measurable neMDH presence could be detected via PCR, gene-specific primers detected two 1 kb amplicons of cMDH, designated LangMDH1 (corresponding to +P, HQ690186) and LangMDH2 (corresponding to −P, HQ690187), respectively. Sequencing analyses of these cMDH amplicons showed them to be 96% identical on an amino acid level. There was a high degree of diversification between proteins detected in this study and other known MDH proteins, particularly those from other leguminous plants. Enhanced malate synthesis in P-deficient nodules was achieved via increased anaplerotic CO2 fixation and subsequent higher MDH activities. Novel isoforms of cytosolic MDH may be involved, as shown by gene expression of specific genes under P deficiency.