Accumulating studies have demonstrated that drug-resistance remains a great obstacle for the effective treatment of cancers. Esophageal cancer is still one of the most common cancers worldwide, which also suffers from the drug-resistance during clinical treatment. Here we performed drug-resistance profiling assays and identified several drug-resistant and drug-sensitive esophageal cancer cell lines. The following methylation sequencing showed that the MCTP1 gene is hypermethylated in the drug-resistant esophageal cancer cells. As a result, the expression of MCTP1 is down-regulated in the drug-resistant esophageal cancer cells. Down-regulation of MCTP1 also affects the migration and apoptosis of esophageal cancer cells, as revealed by the wound-healing and apoptosis assays. Further investigations proposed two signaling pathways that might involve in the MCTP1-mediated drug-resistance of esophageal cancer cells. All these results suggested that MCTP1 activates the drug-resistance of esophageal cancer cells, which has implications for further design of new biomarker of esophageal cancer treatment.