When aurintricarboxylic acid (ATA) was added at a concentration of 30 mg/l in DME/F12 medium to Chinese hamster ovary (CHO) NTHU-108 cells in static six-well plates, some of the cells exhibited adherent growth while others grew in suspension. Beyond the critical concentration of ATA, CHO cells grew in single-cell suspension. In the same serum-free medium, insulin at a level of 0.5 mg/l was found to be the most important protein ingredient promoting cell growth. We used the respective kinase inhibitors to investigate their influence on the cell proliferation induced by ATA and insulin. It is interesting that the inhibition by seven kinase inhibitors of ATA-induced proliferation is similar to that of insulin-induced proliferation. It is possible that ATA mimics insulin and influences the mitogen activated signal transduction to induce the proliferation of CHO cells. Although the actual mechanism of the proliferation of CHO cells by ATA is unclear, ATA supported the long-term proliferation of CHO cells under serum-free conditions and thus could be used as a good substitute for insulin in the formulation of protein-free media.