Previous studies have shown that the expression of claudin-4 is upregulated in breast cancer. The aim of the present study was to investigate the role and the regulation of claudin-4 in MCF-7 breast cancer cells. For the in vitro experiments, MCF-7 cells were treated with recombinant vectors carrying cDNA for claudin-4 overexpression or short hairpin RNAs (shRNAs) for claudin-4 silencing. Cell proliferation was determined by an MTT assay and cell migration ability was measured by a wound-healing assay. The cell cycle profile and apoptotic rate were analyzed using flow cytometry. The effect of methylation status on claudin-4 expression was determined by PCR and western blotting. For the in vivo tumorigenesis analysis, MCF-7 cells with or without claudin-4 silencing were transplanted into nude mice. In vivo cell growth was evaluated 14 days after transplantation. We found that claudin-4 overexpression increased MCF-7 cell proliferation and migration, and reduced the rate of cell apoptosis. Silencing of claudin-4 induced the opposite effects in MCF-7 cells. In addition, claudin-4 expression was upregulated by demethylation. Moreover, the size of tumor formation was reduced in nude mice transplanted with claudin-4 silenced MCF-7 cells. These observations suggested that claudin-4, which was regulated by methylation status, plays an important role in breast cancer growth and malignancy via the control of cell proliferation, migration and apoptosis.