Microbial oil has lots of similarity to the plant’s oil; the only difference between them is that microbial oil is rich in unsaturated fatty acids. This oil can be used in pharmaceutical industry for technical purposes or as edible oil in food industry. The purpose of this study was to increase lipid production and the amount of unsaturated fatty acids in oleaginous yeasts by mutagenesis. In this study, optimization of lipid production in the yeast, Yarrowia lipolytica, was done using ultraviolet random mutation and screening was done with the inhibitory effect of cerulenin on lipid synthesis. Mutants that had normal growth in the presence of cerulenin were selected as superior strains for further investigations. Lipid production in mutants and wild strains were compared after incubation period. Then, the mutant with significant increase in lipid production was selected. The best mutant had increasing of 50% and 30% in lipid production and growth yield, respectively. Also, Linoleic acid was increased after mutation. The composition of fatty acids in the lipid profile of the evaluated yeast did not change a lot after mutation. Of course, unsaturated fatty acids were increased, so this technique was very effective to improve lipid production. Cerulenin used as an effective screening technique in this study and paved the way for the isolation of superior mutants.