Radiotherapy is a common therapeutic strategy used to treat esophageal squamous cell carcinoma (ESCC). However, tumor cells often develop radioresistance, thereby reducing treatment efficacy. Here, we aimed to identify the mechanisms through which ESCC cells develop radioresistance and identify associated biomarkers. Eca109 cells were exposed to repeated radiation at 2 Gy/fraction for a total dose of 60 Gy (Eca109R60/2Gy cells). MTT and colony formation assays were performed to measure cell proliferation and compare the radiation biology parameters of Eca109 and Eca109R60/2Gy cells. Cell cycle distributions and apoptosis were assessed by flow cytometry. Reverse transcription-quantitative polymerase chain reaction and western blotting were employed to analyze the expression of HOX transcript antisense RNA (HOTAIR), in addition to biomarkers of the epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs). Eca109R60/2Gy cells exhibited increased cell proliferation and clone formation, with significantly higher radiobiological parameters compared with the parental Eca109 cells. The Eca109R60/2Gy cells also exhibited significantly decreased accumulation in G(2) phase and increased accumulation in S phase. Additionally, the apoptosis rate was significantly lower in Eca109R60/2Gy cells than in parental Eca109 cells. Finally, HOTAIR expression levels and SNAI1 and beta-catenin mRNA and protein expression levels were significantly higher, whereas E-cadherin levels were significantly lower in Eca109R60/2Gy cells than in Eca109 cells. Therefore, our findings demonstrated that radioresistance was affected by the expression of HOTAIR and biomarkers of the EMT and CSCs.