Abstract：Microbial oil production is a feasible source of raw materials for biodiesel industry. Because of its excellent capability in grease synthesis and accumulation, Rhodotorula glutinis is a good candidate to produce microbial oil by molasses fermentation. The influencing factors, such as culture volume, pH value, inoculum concentration, temperature, nitrogen and carbon nutrition, were investigated by batch cultures. The results indicate that a dry-cell yield of 2.42g/L and a grease yield of 0.8g/L are obtained after fermenting for 132h with an initial COD concentration of 11100mg/L, pH value of 6.5, temperature of 30℃ and swing at 160r/min. The corresponding specific dry-cell and grease yield by COD removal are 0.35 and 0.12kg/kg, respectively. The dry-cell yield and grease yield could be increased to 3.74 and 1.31g/L, respectively, by adding 10g/L glucose instantly at a certain fermentation moment. The addition of nitrogenous nutriments to the fermentation system could promote the multiplication of R. glutinis remarkably, but has no observable influence on the grease yield.