No effective biomarkers have been confirmed to predict chemosensitivity for patients with gastric cancer. The purpose of this study was to investigate whether DNA methylation is associated with chemosensitivity in patients with gastric cancer. Tumors and matched non-tumor biopsy tissues collected from 134 advanced gastric cancer (AGC) patients prior to fluorouracil-based chemotherapy were retrospectively analyzed. The methylation status of p16, E-cadherin (CDH1), MGMT (O-6-methylguanine-DNA methyltransferase), and human mutL homolog 1 (hMLH1) was evaluated using a Methylight assay, and the association between p16 methylation and the sensitivity of 5-fluorouracil in cell lines was determined by in vitro assay. The methylation of p16 (17.9 vs. 0 %, P = 0.002), CDH1 (20.9 vs. 2.2 %, P < 0.001), MGMT (17.9 vs. 0 %, P = 0.052), and hMLH1 (14.9 vs. 2.2 %, P = 0.024) was more common in gastric cancer tissues (n = 134) than in non-tumor tissues (n = 46). For all patients, a reverse correlation was only found between p16 methylation and clinical response (P = 0.017), which suggested that p16 methylation might be associated with chemosensitivity of fluorouracil in gastric cancer patients. Results from in vitro experiments demonstrated that p16 methylation was closely correlated with the sensitivity of 5-fluorouracil in gastric cancer cells. The present results indicated that the methylation of p16, CDH1, MGMT, and hMLH1 was both frequent and specific in gastric cancer tissues. p16 Methylation might be used to predict chemosensitivity of fluorouracil for patients with AGC when validated in large samples in the future.