Abstract: In fed-batch cultivation of recombinant Pichia pastoris (Mut~S phenotype), feeding with glycerol and methanol was conducted during the expression phase to enhance the cell growth and angiostatin expression. The cell density reached 174 g/L at the end of fermentation, which was about 3 fold of that obtained with methanol as the sole carbon source in the induction phase. High cell density resulted in a quick drop of ammonium concentration in the fermentation broth, and when the ammonium concentration was below 40 mmol/L, the production of angiostatin also decreased. The change of pH control strategy and addition of 25 mmol/L (NH_4)_2SO_4 were applied to release the effect caused by low ammonium concentration. The ammonium concentration was maintained above 150 mmol/L in the induction phase, and 108 mg/L angiostatin was achieved at the end of fermentation.