Oleaginous microorganisms are defined as oleaginous species with oil contents excess of 20% of biomass weight ,Rhototorula glutinis is an Oleaginous yeast ,which is able to accumulate great amount of total lipid. The comparison of a 15 L agitation tank and a 50 L airlift bioreactor on the growth of R.glutinis and total lipid production by batch operation was studied, and the effects of three fed-batch feeding criterias on the growth and lipid production were also examined. Results indicated that operation in a 50 L airlift bioreactor had superior performance on the growth of R.glutinis than that of the 15 L agitation tank. The fed-batch operation could provide sufficient carbon source-crude glycerol while avoiding inhibition resulting from the accumulation of high crude glycerol. Therefore, fed-batch operation could promote a high cell density as compared to that of batch operation. Among the feeding criteria, the exponential feeding criteria could avoid the accumulation of high crude glycerol concentration, and led to the enhancement of cell growth rate. Nevertheless, the conventional pulse feeding criteria could achieve the highest total lipid and β-carotene productivity. Consequently, if collecting the maximum total lipids for biodiesel production is the goal, the fed-batch operation by pulse feeding is suggested as a suitable criterion for the cultivation of R.glutinis with crude glycerol as the carbon source.