Ras association domain family 1A (RASSF1A) gene inactivation by promoter hypermethylation is a common event in the development of a variety of types of human cancer. Accumulated evidence has demonstrated that DNA methylation serve a critical role in the pathogenesis of endometriosis. The aim of the present study was to analyze the methylation status and protein expression of RASSF1A in endometriosis (EMS). The ectopic and corresponding eutopic endometrium tissues were collected from 45 women with EMS (EMS group) and normal endometrium tissues from 20 women without EMS (control group). The methylation status of RASSF1A was examined by methylation specific polymerase chain reaction (MSP). Immunohistochemistry was performed to measure RASSF1A protein level in endometrium tissues. In normal endometrium samples, RASSF1A protein expression was significantly higher at the secretory phase than the proliferative phase. RASSF1A protein expression in the ectopic endometrium tissues and eutopic endometrium tissues were significantly reduced than in normal endometrium (P<0.05). The frequency of aberrant methylation of RASSF1A was 55.56% in ectopic endometrium and 33.33% in paired eutopic endometrium, whereas such methylation was not detected in normal endometrium. Moreover, RASSF1A promoter hypermethylation was frequently associated with reduced expression of RASSF1A, and was common in advanced stage in ectopic endometrium of EMS. Epigenetic inactivation of RASSF1A through aberrant promoter methylation may be important in the formation and progression of EMS, and assessment of RASSF1A methylation status in eutopic endometrium may be a potentially useful biomarker to enhance the early detection of EMS.