This study demonstrates the potential of the oleaginous fungus Mucor circinelloides for synthesizing oil rich in polyunsaturated fatty acids when cultivated in thin stillage (TS) and centrifuged thin stillage (CTS) from corn-to-ethanol production. Biomass yield, oil yield and its EPA content were studied at different cultivation periods and carbon to nitrogen ratios. The fungal biomass yield (g/L) and its oil content (g/L) were found to be twice as high in TS as in CTS when harvested after two to six days. EPA content doubled in TS and increased by 2.6 times in CTS when the cultivation period was extended from three to six days. Glycerol supplementation at 20g/L of C led to an 18% increase and 63.6% decrease in fungal oil yield in TS and CTS respectively. Glycerol addition also increased EPA content by 33% in TS and 60% in CTS. Adding 5g/L N using ammonium sulfate as a nitrogen source enhanced the fungal oil yield by 34% in TS and 26% in CTS and EPA content by 32% in TS and 12% in CTS. Urea at 0.5 g/L of N increased the EPA content by 22% in TS and 33% in CTS and oil content by 12% in TS and 13% in CTS. Potassium nitrate at 5g/L of N increased EPA content by 28% in TS and 33% in CTS at 0.5 g/L of N dosage and increase in oil yield by 18% in TS and decreased oil yield in CTS by 37%.