In the present study, the yeast Rhodotorula glutinis has been assessed with the aim of producing microbial lipids from glycerol under different aeration conditions. For such a purpose, experiments were carried out in shake flasks, under different conditions of agitation (150 to 250 rpm) and aeration (2.5 to 5.0 of flask volume-to-medium volume ratio). Furthermore, their influence on fermentative parameters (lipid and cell concentration, biomass yield; lipid yield; and lipid volumetric productivity) has been investigated using a 2 2 full factorial design. The statistical analysis has revealed a strong influence of both variables on substrate consumption, lipid accumulation, cell growth and lipid productivity. As a whole, results suggest that higher aeration levels provide greater cell and lipid concentrations, and lipid volumetric productivity. The best results (4.5 g/L of lipids and Q P = 0.95 g/L•day) were achieved at the highest aeration (5.0 flask volume-to-medium volume ratio) and agitation (250 rpm) levels. Their fatty acid profile showed that oleic acid was produced in greater quantity (53.5%), followed by linoleic acid (18.7%), palmitic acid (6.8%) and stearic acid (9.9%). The microbial oil presented viscosity of 39.3 cP at 50˚C and free fatty acid content of 1.93% ± 0.08%. These are significant results and contribute to establishing operational conditions that maximize single-cell oil production from glycerol by Rhodotorula glutinis, i.e. an alternative source as renewable raw material for lipid-based biorefineries.