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Background and Aims: Esophageal squamous cell carcinoma (ESCC), a major histologic subtype of esophageal cancer, is increasing in incidence, but the genetic underpinnings of this disease remain unexplored. The aim of this study is to identify the recurrent genetic changes, elucidate their roles and discover new biomarkers for improving clinical management of ESCC. Methods: Western blotting and immunohistochemistry were performed to detect the expression level of RHCG. Bisulfite genomic sequencing (BGS) and methylation-specific PCR (MSP) were used to study the methylation status in the promoter region of RHCG. The tumor-suppressive effect of RHCG was determined by both in-vitro and in-vivo assays. Affymetrix cDNA microarray was used to identify the underlying molecular mechanism. Results: RHCG was frequently downregulated in ESCCs, which was significantly correlated with poor differentiation (P = 0.001), invasion (P = 0.003), lymph node metastasis (P = 0.038) and poorer prognosis (P < 0.001). Demethylation treatment and bisulfite genomic sequencing analyses revealed that the downregulation of RHCG in both ESCC cell lines and clinical samples was associated with its promoter hypermethylation. Functional assays demonstrated that RHCG could inhibit clonogenicity, cell motility, tumor formation and metastasis in mice. Further study revealed that RHCG could stabilize IκB by decreasing its phosphorylation, and subsequently inhibit NF-κB/p65 activation by blocking the nuclear translocation of p65, where it acted as a transcription regulator for the upregulation of MMP1 expression. Conclusions: Our results support the notion that RHCG is a novel tumor suppressor gene that plays an important role in the development and progression of ESCC.