Molecular iodine (I₂) is known to inhibit the induction and promotion of N-methyl-n-nitrosourea-induced mammary carcinogenesis, regresses 7, 12-Dimethylbenz (a)-anthracene-induced breast tumors in rat and has also shown beneficial effect in the fibrocystic human breast disease. Cytotoxicity of iodine on cultured human breast cancer cell lines viz. MCF-7, MDA-MB-231, MDA-MB-453, ZR-75-1 and T-47D is reported in this communication. Iodine induces apoptosis in all the cell lines tested, except MDA-MB-231 shown by sub-G1 peak analysis using flow cytometry. Iodine inhibited proliferation of normal human peripheral blood mononuclear cells, however did not induce apoptosis in these cells. Iodine-induced apoptotic mechanism was studied in MCF-7 cells. DNA fragmentation analysis confirmed internucleosomal DNA degradation. Terminal deoxynucleotidyl transferase-dUTP nick end labeling established that iodine induces apoptosis in time and dose- dependent manner in MCF-7 cells. Iodine-induced apoptosis is independent of caspases. Iodine dissipates mitochondrial membrane potential, exhibits an antioxidant activity and causes depletion in total cellular thiol content. Western blot results showed decrease in Bcl-2 and upregulation of Bax. Immunofluorescence studies confirmed activation and mitochondrial membrane localization of Bax. Ectopic Bcl-2 overexpression did not rescue iodine-induced cell death. Iodine treatment induces translocation of Apoptosis inducing factor from mitochondria to the nucleus. Treatment of N-acetyl-L-cysteine prior to iodine exposure restored basal thiol content, ROS levels and completely inhibited nuclear translocation of Apoptosis inducing factor and subsequently cell death, indicating that thiol depletion may play an important role in iodine-induced cell death. These results demonstrate that iodine treatment activates a caspase-independent and mitochondria-mediated apoptotic pathway.