To investigate the possible mechanism of apoptosis induced by indole-3-acetic acid (IAA) combined with horseradish peroxidase in leukemia cell line K562, cell proliferation and apoptosis of K562 cell were examined by MTT assay and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), respectively; the activity of superoxide dismutase (SOD) and the quantitative change of MDA were measured by biochemical method; changes of free radical were determined by 2, 7-dichlorofluorescin diacetate (DCFH-DA) probe with confocal microscopy. The results showed that of MTT assay and TUNEL indicated that IAA/HRP could significantly inhibit cell proliferation (P < 0.05) and induce apoptosis of K562 cell (P < 0.01), at the same time a positive correlation was found between apoptosis rate and IAA concentration (r = 0.971, P < 0.01). The activity of SOD and the quantitative of MDA increased, accompanied with a rise in IAA concentration. Results detected by DCFH-DA probe indicated that the fluorescence intensity of intracellular free radical increased, as compared with control, and a positive correlation was found. It is concluded that IAA/HRP can inhibit proliferation of K562 cells and induce K562 cell apoptosis, its mechanism may be related with the increase of intracellular free radical due to the effects of IAA/HRP.