14-3-3σ proteins regulate numerous cellular processes that are important to cancer development. One of its biological roles involves G2 cell-cycle arrest following DNA damage. It has also been reported that the loss of 14-3-3σ expression via CpG methylation may contribute to malignant transformation by impairing the G2 cell-cycle checkpoint function, thereby allowing an accumulation of genetic defects. However, how the CpG methylation-dependent silencing mechanism works in relation to promoter methylation associated with methyl-CpG-binding proteins (MeCPs) is still unclear. To better understand the mechanism, we First examined the methylation status of the 14-3-3σ promoter-associated CpG islands and 14-3-3σ gene expression in a subset of prostate cancer cell lines using methylation-specific PCR (MSP), an HhaI-based DNA methylation assay, and reverse transcription-PCR (RT-PCR). We found that the 14-3-3σ expression is lost in LNCaP and Tramp-C1 prostate cancer cell lines and that this expression is restored after treatment with epigenetic silencing modifiers 5-aza-2′-deoxycytidine (5-aza) and trichostatin A (TSA). These results imply transcriptional silencing via promoter-associated CpG methylation. Chromatin immunoprecipitation analysis revealed that methyl-CpG-binding protein 2 (MBD2) is associated preferentially to the methylated CpG island in the 14-3-3σ promoter in LNCaP and Tramp-C1 cells but not in 14-3-3σ-expressing PC3 and DU145 cells, which contain an unmethylated CpG island in the 14-3-3σ promoter region. The 14-3-3σ gene silencing because of CpG methylation correlates with binding of MBD2. In addition, the activation of 14-3-3σ gene expression by a combination of 5-aza and TSA also involves the release of the MBD2 from the 14-3-3σ promoter-methylated CpG island in LNCaP and Tramp-C1 cells. Furthermore, MBD2 knockdown by siRNA stimulated 14-3-3σ expression in LNCaP cells. We also investigated whether the loss of 14-3-3σ expression in LNCaP and Tramp-C1 cells affects cell proliferation by MTT assays. Interestingly, we observed that 14-3-3σ-inactivated LNCaP and Tramp-C1 cells had markedly decreased cell proliferation and protein expression of proliferation cell nuclear antigen (PCNA) after restoration of 14-3-3σ expression with 5-aza and TSA treatment. On the other hand, the same treatment did not significantly affect 14-3-3σ-active PC3 and DU145 cells, which normally express 14-3-3σ. Finally, 14-3-3σ knockdown by siRNA resulted in increased proliferation in PC3 and DU145 cells. These findings suggest that the transcriptional silencing of the 14-3-3σ gene is caused by promoter CpG island methylation associated with MBD2, and that this may play an important role in prostate cancer progression during the invasive and metastatic stages of the disease.