The protein complex S100A8/A9, abundant in the cytosol of neutrophils, is secreted from the cells upon cellular activation and induces apoptosis in tumor cell lines and normal fibroblasts in a zinc‐reversible manner. In the present study, we present evidence that the S100A8/A9 also exerts its apoptotic effect by a zinc‐independent mechanism. Treatment of the colon carcinoma cells with different concentrations of human S100A8/A9 or the metal ion chelator diethylenetriaminepentacetic acid (DTPA) resulted in a significant increase of cell death. Annexin V/phosphatidylinositol and Hoechst 33258 staining revealed that cell death was mainly of the apoptotic type. A significant increase in the activity of caspase‐3 and ‐9 was observed in both cell lines after treatment. Caspase‐8 activation was negligible in both cell lines. The cytotoxicity/apoptotic effect of human S100A8/A9 and DTPA was inhibited significantly (P<0.05) by Zn+2 and Cu+2, more effectively than by Ca2+ and Mg2+. The antioxidant N‐acetyl‐L‐cysteine inhibited the cytotoxicity/apoptotic effect of S100A8/A9 and DTPA. However, as a result of the different time‐courses of both agents and that the S100A8/A9‐induced apoptosis was not completely reversed, we conclude that S100A8/A9 exerts its apoptotic effect on two colon carcinoma cell lines through a dual mechanism: one via zinc exclusion from the target cells and the other through a yet‐undefined mechanism, probably relaying on the cell‐surface receptor(s).