Repeated addition of insulin, together with an initial addition of the reduced form of glutathione (GSH) and a chelator, aurintricarboxylic acid (ATA), was proposed as a strategy for the dynamic control of apoptosis in serum-free culture of Chinese hamster ovary (CHO) cells. Insulin suppressed cell death in serum-free culture by means of lowering intracellular reactive oxygen species levels via an increase in the specific glucose consumption rate. This effect of insulin addition was found not only at 0 h but also at 48 h during serum-free cultivation. In consideration of the reduction of insulin by GSH, insulin was again added at 24 h following the initial addition during the serum-free cultivation. The repeated addition of insulin decreased cell death and increased the production of tissue plasminogen activator compared with the single addition at 0 h.