Raf kinase inhibitory protein (RKIP) regulates multiple cellular processes, and its downregulation is associated with distinct human cancers. In the present study, the status of RKIP promoter methylation, as well as its expression and clinical significance in esophageal squamous cell carcinoma (ESCC), were examined. The promoter methylation status in the 5'-CpG island of the RKIP gene and the expression level of the RKIP protein were examined using a modified methylation-specific polymerase chain reaction (MSP) method and immunohistochemical staining, respectively, in 77 ESCC samples and matched paratumor normal tissues. The incidence of RKIP promoter methylation was significantly higher in tumor samples (75.3%) than in the matched normal tissues (27.3%; P < 0.001). A higher incidence of promoter methylation was also detected in poorly differentiated cancers (93.5%) compared with well-differentiated cancers (50.0%; P < 0.001), as well as in tumor samples with positive lymph node metastasis (86.7%) compared with those with negative lymph node metastasis (59.4%; P < 0.001). Consistent with the promoter methylation status, the expression level of RKIP was significantly reduced in cancer tissues (36.4%) compared with matched normal tissues (76.6%; P < 0.01), as well as in cancers with positive lymph node metastasis (24.4%) compared with those with negative lymph node metastasis (53.1%; P=0.01). Promoter methylation-induced gene silencing significantly correlated with the down regulation of RKIP and the development of ESCC. The results of the present study suggested that the methylation status of the RKIP promoter, when combined with its expression level, may serve as a biomarker for predicting the biological behaviors of ESCC.