Purpose: In this study, we examined the promoter methylation status and expression of 14-3-3σ and evaluated its clinical significance in epithelial ovarian cancer. Experimental Design: Twelve ovarian cancer cell lines; 2 ovarian surface epithelial cell lines; and 8 normal, 8 benign, 12 borderline, and 102 ovarian cancer tissues were examined. Methylation-specific PCR, quantitative reverse transcription-PCR, and immunohistochemistry were used to evaluate methylation status and expression of 14-3-3σ gene and protein. Results: Among the 12 ovarian cancer cell lines, the presence of a methylated band was detected in seven cell lines. Median values of relative 14-3-3σ gene expression in cancers with methylation (3.27) were significantly lower than those without methylation (16.4; P < 0.001). Treatment of 5-aza-2′-deoxycitidine resulted in the demethylation of the promoter CpG islands and reexpression. All of the normal, benign, and borderline tissues were positive for 14-3-3σ protein, and in ovarian cancer tissues, 73.5% (75 of 102) were positive for 14-3-3σ protein and was almost consistent with methylation status. Negative immunoreactivity of 14-3-3σ was significantly correlated with high age and serous histology, high-grade, advanced-stage residual tumor of >2 cm, high serum CA125, high Ki-67 labeling index, and positive p53 immunoreactivity. 14-3-3σ immunoreactivity was significantly associated with overall survival (P = 0.0058). Conclusions: Our findings suggest that 14-3-3σ is inactivated mainly by aberrant DNA methylation and that it may play an important role in the pathogenesis of epithelial ovarian cancer.