3,3'-Diindolylmethane (DIM) is a potential cancer preventive phytochemical derived from Brassica vegetables. The effects of DIM on cell-cycle regulation in both estrogen-dependent MCF-7 and estrogen receptor negative p53 mutant MDA-MB-468 human breast cancer cells were assessed in this study. DIM inhibited the breast cancer cell growth in vitro and in vivo, and caused cell-cycle arrest by down-regulating protein levels of cell-cycle related kinases CDK1, CDK2, CDK4, and CDK6, as well as Cyclin B1 and Cdc25A. Meanwhile, it was revealed that Ser(124) phosphorylation of Cdc25A is primarily responsible for the DIM-induced Cdc25A degradation. Furthermore, treatment of MCF-7 cells with DIM increased miR-21 expression and down-regulated Cdc25A, resulting in an inhibition of breast cancer cell proliferation. These observations collectively suggest that by differentially modulating cellular signaling pathways DIM is able to arrest the cell-cycle progression of human breast cancer cells.