The oleaginous yeast Yarrowia lipolytica has considerable potential for producing single cell oil, which can be converted to biodiesel, a sustainable alternative to fossil fuels. However, extensive fundamental and engineering efforts must be carried out before commercialized production become cost-effective. Therefore, in this study, metabolic flux analysis of Y. lipolytica was performed using 13C-labeled glucose as a sole carbon source in nitrogen sufficient and insufficient media. The nitrogen limited medium inhibited cell growth while promoting lipid accumulation (from 8.7% of their biomass to 14.3%). Metabolic flux analysis showed that flux through the pentose phosphate pathway was not significantly regulated by nitrogen concentration, suggesting that NADPH generation is not the limiting factor for lipid accumulation in Y. lipolytica. Furthermore, metabolic flux through malic enzyme was undetectable, confirming its non-regulatory role in lipid accumulation in this yeast. Nitrogen limitation significantly increased flux through ATP:citrate lyase (ACL), implying that ACL plays a key role in providing acetyl-CoA for lipid accumulation in Y. lipolytica.