Early biochemical studies have demonstrated that lipid accumulation by oleaginous yeasts is linked to the activity of the NAD(+)-dependent isocitrate dehydrogenase (Idh). However, molecular study of Idh of oleaginous microorganisms remains limited. Here, we present the cloning of a mitochondrial NAD(+)-specific Idh from Rhodosporidium toruloides (RtIdh), an excellent microbial lipid producer that uses carbohydrates as the carbon source. The evolutionary relationship analyses among RtIdhs and other yeast Idhs revealed that RtIdh had a closer relationship with the Idhs of Ustilago maydis and Schizophyllum commune. We expressed the RtIDH gene in the yeast Saccharomyces cerevisiae idh Delta mutant. Under the nitrogen-limited condition, the intracellular lipid content and extracellular citrate concentration of the culture of the S. cerevisiae idh Delta carrying the RtIDH gene increased as the carbon/nitrogen molar ratio of the media increased, while the wild-type S. cerevisiae strain showed no correlation. Our data provided valuable information for elucidating the molecular mechanism of microbial oleaginicity and for engineering microorganisms to produce metabolites of fatty acid pathway.