In recombinant Pichia pastoris fermentation for hirudin production, copious cells were not viable and most of the secreted hirudin molecules were C-terminally truncated at the end of fermentation. In this work, the influences of reactive oxygen species (ROS) on cell viability and hirudin production were subsequently studied. In contrast to the untreated control condition, the addition of ascorbic acid at the methanol fed-batch phase could obviously relieve the damage of intracellular ROS to cell membranes. As a result, the cell viability could be increased to 91% from 74% in control at the end of fermentation and the extracellular proteolysis of hirudin reduced. Intact and total hirudin production, by supplying ascorbic acid, could reach 2.90 and 5.03 g/l, respectively, in contrast to 1.75 and 4.70 g/l at the control condition. Ascorbic acid, 4 mmol/l or more, in the fermentation broth increased markedly the production of the intact hirudin, despite a little effect on total hirudin production.