The nel-like1 (NELL1) gene maps to chromosome 11p15, which frequently undergoes loss of heterozygosity in esophageal adenocarcinoma (EAC). NELL1 promoter hypermethylation was examined by real-time methylation-specific polymerase chain reaction in 259 human esophageal tissues. Hypermethylation of this promoter showed highly discriminative receiver–operator characteristic curve profiles, clearly distinguishing esophageal squamous cell carcinoma (ESCC) and EAC from normal esophagus (NE)(P< 0.001). NELL1 normalized methylation values were significantly higher in Barrett's metaplasia (BE), dysplastic Barrett's (D) and EAC than in NE (P< 0.0000001). NELL1 hypermethylation frequency was zero in NE but increased early during neoplastic progression, to 41.7% in BE from patients with Barrett's alone, 52.5% in D and 47.8% in EAC. There was a significant correlation between NELL1 hypermethylation and BE segment length. Three (11.5%) of 26 ESCCs exhibited NELL1 hypermethylation. Survival correlated inversely with NELL1 hypermethylation in patients with stages I–II (P= 0.0264) but not in stages III–IV (P= 0.68) EAC. Treatment of KYSE220 ESCC and BIC EAC cells with 5-aza-2′-deoxycytidine reduced NELL1 methylation and increased NELL1 mRNA expression. NELL1 mRNA levels in EACs with an unmethylated NELL1 promoter were significantly higher than those in EACs with a methylated promoter (P= 0.02). Promoter hypermethylation of NELL1 is a common, tissue-specific event in human EAC, occurs early during Barrett's-associated esophageal neoplastic progression, and is a potential biomarker of poor prognosis in early-stage EAC.