Cancer-associated fibroblasts (CAFs), as one major component of tumor microenvironment (TME), are closely associated with tumor initiation and progression. Our previous studies have discovered that CAFs induced the resistance of esophageal squamous cell carcinoma (ESCC) cells to a variety of chemotherapeutic drugs such as cisplatin and paclitaxel. Furthermore, CAFs attenuated ionizing irradiation (IR)-induced cancer cells death by regulating DNA damage response. CAFs themselves are highly resistant to death stimuli due to enhanced antioxidant potential and DNA repair capacity. How to inhibit the malignant phenotype of CAFs is critically important for the radical treatment of ESCC. By RNA-sequencing and DNA methylation analysis, the transcriptome and epigenome of CAFs and matched normal fibroblasts (NFs) have been integratively analyzed. By transfection of TCF21 cDNA plasmid, the expression of TCF21 in CAFs has been up-regulated. Using a cell counting kit and migration and invasion assay, the effect of TCF21 on the growth and migration and invasive ability of CAFs has been detected. Using immunofluorescence and flow cytometry (FCM) analysis and western blotting, the effect of TCF21 on the DNA damage repair and apoptotic death of CAFs following IR has been detected. TCF21 is one of the top ten down-regulated genes in CAFs compared with NFs due to promoter methylation. Up-regulation of TCF21 expression inhibited the growth rate and migration and invasive ability of CAFs. The expression of α-SMA, as an indicator of CAFs activation, was down-regulated in CAFs which were transfected with TCF21 cDNA. Furthermore, when TCF21 cDNA was transfected into CAFs, IR-induced DNA damage was increased while DNA repair was inhibited in CAFs, suggesting that TCF21 was involved in DNA damage response of CAFs following IR. FCM analysis showed that up-regulation of TCF21 expression promoted IR-induced apoptotic death of CAFs. TCF21 is a determinant of the malignant phenotype of CAFs in ESCC. Up-regulation of TCF21 expression is a promising approach of inhibiting the growth, migration and invasion, activation and radioresistance of CAFs in ESCC.