Esophageal squamous cell carcinoma (ESCC), the most common esophageal cancer in East Asia, is among the six cancers with the highest fatality rates worldwide. Unfortunately, multidisciplinary treatment strategies have not achieved satisfactory outcomes. Therefore, novel insights into the molecular biology of ESCC are required to improve treatment. The gene encoding the transmembrane adherens junctions-associated protein-1 (AJAP1) expressed by epithelial cells resides in chromosome 1p36, which is frequently lost or epigenetically silenced in several malignancies. Here, we investigated the expression levels and regulatory mechanism of AJAP1 transcription. We determined the levels of AJAP1 mRNA and the genes encoding potentially interacting proteins expressed by ESCC cell lines, as well as the chromosomal copy number of AJAP1 and the methylation status of its promoter region. AJAP1 mRNA levels of 78 pairs of surgically resected specimens were determined to evaluate the association of AJAP1 expression and clinicopathological factors. Nine ESCC cell lines differentially expressed AJAP1 mRNA, and demethylation of hypermethylated AJAP1 genomic DNA reactivated AJAP1 mRNA expression. The copy number of sequences upstream or downstream of the AJAP1 transcriptional start site was not detectably altered. AJAP1 mRNA levels correlated inversely with those of ezrin (EZR) and were significantly lower in ESCC tissues compared with adjacent normal tissues. AJAP1 mRNA levels decreased gradually with increasing tumor stage. Patients with downregulated AJAP1 transcription were more likely to experience shorter overall and disease-free survival. Multivariate analysis of disease-free survival identified downregulated AJAP1 transcription as an independent prognostic factor. These results suggest that in ESCC, AJAP1 acts as a putative tumor suppressor and that AJAP1 transcription is regulated by promoter hypermethylation. These findings indicate that downregulated AJAP1 transcription may serve as a novel tumor biomarker to predict recurrence of ESCC after esophagectomy.