Objective: To detect promoter region methylation of Fibulin-1 gene in esophageal cancer and explore its clinical significance. Methods: Ninety-six patients of esophageal cancer admitted from January 2014 to February 2016 in Huangshi Central Hospital were enrolled in this study. Another 40 cases of benign esophageal tissues were used as control. Fibulin-1 gene promoter was modified by bisulfite and methylation was detected using PCR analysis. Fibulin-1 protein expression was detected by immunohistochemistry. Results: The positive rate of Fibulin-1 gene promoter methylation in patients with esophageal cancer was 36.5%, which was significantly higher than that of 15.0% in control group (χ2=2.517, P=0.036). The positive expression rate of Fibulin-1 protein in the promoter methylation of Fibulin-1 gene was 90.2%. The positive rate of Fibulin-1 gene promoter methylation has no significant difference in different gender and age in bladder cancer (P>0.05). The positive rate of Fibulin-1 gene promoter methylation in Ⅲ and Ⅳ stage group was 43.1%, which was higher than that of 28.9% in Ⅰ and Ⅱ stage group (χ2=2.426, P=0.046). The positive rate of Fibulin-1 gene promoter methylation in lymph node metastasis group was 44.4%, which was higher than that of 26.2% in non-lymph node metastasis group (χ2=2.438, P=0.041). The positive rate of Fibulin-1 gene promoter methylation in median and low differentiation group was 42.6%, which was higher than that of 25.7% in well-differentiation group (χ2=2.431, P=0.043). Conclusion: Hypermethylation of Fibulin-1 gene was detected in esophageal cancer, which was related to clinical stages, lymph node metastasis, and tumor differentiation of esophageal cancer.