The oleaginous yeast Lipomyces starkeyi can accumulate intracellular lipids to over 60% of its cell dry mass under a nitrogen-limited condition. We showed that extracellular and intracellular citrate concentrations of L. starkeyi AS 2.1560 increased and the nicotinamide adenine dinucleotide - isocitrate dehydrogenase (NAD+-IDH) activity decreased at the beginning of the lipid accumulation, suggesting that the attenuation of the NAD+-IDH activity might initiate lipid storage. We next cloned the IDH gene by the methods of degenerate PCR and rapid amplification of cDNA ends. Phylogenetic analyses of the evolutionary relationships among LsIDH1, LsIDH2, and other yeast NAD+-IDHs revealed that the L. starkeyi IDH had a closer relationship with the IDHs of Yarrowia lipolytica. Further real-time PCR analysis showed that the expression levels of both LsIDH1 and LsIDH2 decreased concurrently with the evolution of cellular lipids. Our data should be valuable for understanding the biology of oleaginous yeasts and for further strain engineering of L. starkeyi.