The radioresistance of esophageal squamous cell carcinoma is a great obstacle to treatment. Although it has been demonstrated that microRNA-21 (miR-21) can act as an ‘oncogene’ in esophageal squamous cell carcinoma, its role in radioresistance remains unexplored. The aims of this study were to investigate the role of miR-21 in esophageal squamous carcinoma cells' radioresistance and to identify the possible mechanism. The relatively radioresistant esophageal squamous cancer TE-1 cells (TE-R60) was established by fractionated irradiation. By lentiviral transduction with miRZip-21, the miR-21 expression in TE-1 cells was stably downregulated, which was renamed as ‘anti-miR-21 TE-1 cells.’ The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was knocked down in anti-miR-21 TE-1 cells through short interfering RNA. The expression level of miR-21 and PTEN messenger RNA were measured by quantitative real-time reverse transcription polymerase chain reaction or reverse transcription polymerase chain reaction. The expression level of PTEN, phospho-Akt, and Akt protein were detected by Western blot. Clongenic assay was used to analyze the cells' radiosensitivity. miR-21 was overexpressed, and PTEN was suppressed in established radioresistant TE-R60 cells compared with the parent cells (1.3-fold and 70.83%). The inhibition of miR-21 significantly increased the cells' radiosensitivity (P < 0.05) and the PTEN protein expression (2.3-fold) in TE-1 cells. In addition, phospho-Akt protein, downstream target of PTEN, reduced significantly in anti-miR-21 TE-1 cells. Knockdown of PTEN in anti-miR-21 TE-1 cells could abrogate the miR-21 inhibition-induced radiosensitization (P < 0.05). Inhibition of miR-21 increased radiosensitivity of esophageal cancer TE-1 cells, and this effect was possibly through the activation of PTEN. Inhibition of miR-21 may form a novel therapeutic strategy to increase the radiosensitivity of esophageal cancer.