Yarrowia lipolytica is a potentially useful host for heterologous protein production. To develop an efficient culture method for high cell density cultivation and heterologous gene expression of Y. lipolytica, the effects of medium components and their concentrations on the growth of Y. lipolytica have been investigated. Addition of yeast extract to the culture media was found to significantly reduce the long lag phase encountered when Y. lipolytica was cultivated in synthetic culture media containing high concentrations of glycerol. Therefore, by enriching with 0.3% yeast extract the synthetic culture medium containing 15% glycerol, we could cultivate Y. lipolytica up to 83 g/L dry cell weight in a batch culture. Furthermore, over 100 g/L and 88 units/mL of rice alpha-amylase activity were obtained in less than 50 h with a one-step feeding process in which a recombinant Y. lipolytica expressing rice alpha-amylase was cultivated in the 10% glycerol medium enriched with 0.3% yeast extract and fed only once with the concentrated feeding medium (60% glycerol). The easy cultivation of recombinant Y. lipolytica to a high cell density may strengthen its position as a host for heterologous protein production.