Background: Inactivation of cell-cycle regulating gene p16, resulting from epigenetic alteration, is common in the carcinogenesis of human cancers. The aim of this study is to offer a systematic review on the aberrant methylation of p16 gene in esophageal cancer. Methods: We performed a meta-analysis referring to the guidelines of PRISMA. We searched for articles published from 1996 to 31 May 2012 using PubMed and China National Knowledge Infrastructure (CNKI) database. Additional database including Web of Science and EMBASE were also searched for related articles. The random or fixed effect model was applied to estimate the pooled frequency of DNA methylation based on the heterogeneity analysis. Subgroup analyses were performed according to the histological type, study area, and tumor grade. Results: This meta-analysis included 39 articles related to the methylation studies on p16 gene in cancer tissues and 7 articles using blood samples. The summarized frequency of DNA methylation detected in cancer tissues was 0.53 (95% CI: 0.44–0.61). With the increase of tumor differentiation grades, the frequency of DNA methylation increased accordingly (well differentiated: 0.37; moderately differentiated: 0.61; poorly differentiated: 0.63). We further summarized the methylation of p16 gene detected in patient’s peripheral blood samples. The pooled frequency was 0.33 (95% CI: 0.17–0.49), which was lower than that detected in cancer tissues. Conclusion: This meta-analysis revealed the elevated frequency of DNA methylation of p16 gene in esophageal cancer, which indicated future potential application of this biomarker in early detection as well as the prognosis of the disease.