Natural lipids can be used to make biodiesel and many other value-added compounds. In this work, we explored a number of different metabolic engineering strategies for increasing lipid production in the oleaginous yeast Rhodosporidium toruloides IFO0880. These included increasing the expression of enzymes involved in different aspects of lipid biosynthesis—malic enzyme ( ME ), pyruvate carboxylase ( PYC1 ), glycerol-3-P dehydrogenase ( GPD ), and stearoyl-CoA desaturase ( SCD )—and deleting the gene PEX10 , required for peroxisome biogenesis. Only malic enzyme and stearoyl-CoA desaturase, when overexpressed, were found to significantly increase lipid production. Only stearoyl-CoA desaturase, when overexpressed, further increased lipid production in a strain previously engineered to overexpress acetyl-CoA carboxylase ( ACC1 ) and diacylglycerol acyltransferase ( DGA1 ). Our best strain produced 27.4 g/L lipid with an average productivity of 0.31 g/L/h during batch growth on glucose and 89.4 g/L lipid with an average productivity of 0.61 g/L/h during fed-batch growth on glucose. These results further establish R. toruloides as a platform organism for the production of lipids and potentially other lipid-derived compounds from sugars.