Biodiesel is a useful alternative source of energy and can be used as a support for oil-source diesel. Microbial lipids are considered a promising raw material for biodiesel production. In a previously completed study, Pichia cactophila was found to be the best lipid-producing yeast by lipid screening studies among 107 yeast isolates. In this study, the lipid production conditions of P. cactophila, which was chosen as a lipid producer for the first time, were partially optimized by using response surface methodology in crude glycerol. Plackett-Burman statistical experiment design was used to determine the effect of glycerol, yeast extract, peptone, urea, (NH4)(2)SO4, NH4Cl, NH4NO3 factors on lipid production. In order to determine the optimum levels of the most effective parameters by the result of screening (glycerol, yeast extract, urea, NH4NO3), Box-Behnken experimental design method was used. The optimum production conditions were determined as 39 g/L glycerol, 20 g/L yeast extract, 5 g/L urea, and 20 g/L NH4NO3, and the result of the experiments carried out under these conditions the lipid value (%) was determined as 91.43. Lipid content was increased 1.408-fold (40.79%) by two-step optimization with the response surface methodology and reached a very high value. The fatty acid composition obtained under optimum conditions is compatible with vegetable oils. This suggests that the microbial lipid of P. cactophila can be used as a raw material for biodiesel production and is promising for further studies.