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Recently, Candida antarctica lipase B (Cal B) draws attention from industries for various applications for food, detergent, fine chemical, and biodiesel, because of its characteristics as an efficient biocatalyst. Since many industrial processes carry out in organic solvent and at high temperature, Cal B, which is stable under harsh condition, is in demand from many industries. Cal B was expressed in the methylotrophic yeast Pichia pastoris X-33 and was produced in a large scale fermentor for the commercial application. Using 5L fermenters, cultivation conditions were investigated for the high expression of Cal B in a 500L fermentor. During the cultivation, glycerol and methanol were added to obtain high cell density and for the induction of cal B, respectively. When DO rebounded from the minimum value, the glycerol feeding was started and 8L glycerol was pumped into the fermentor. Thereafter, the methanol feeding was started for the induction of Cal B. After 70 hours cultivation, OD600 for cell density was reached 277, the activity of lipase was 1.5 × 105 U/L, and 2.8 g/L protein was obtained. After the freeze dry, the residual activity of lipase was 5 × 106 U/g.