Esophageal cancer is one of the most common type of malignancies. Telomerase activity, which is absent or weakly detected in the majority of human somatic cells, is elevated in esophageal cancer. Although azidothymidine (AZT), a reverse transcriptase inhibitor, has been utilized as a treatment for tumors, its role in treating esophageal cancer has not been confirmed. The aim of the present study was to determine the effect of AZT on telomerase activity and the proliferation of the human esophageal cancer cell line TE‑11. A telomeric repeat amplification assay was utilized to detect telomerase activity following treatment of TE‑11 cells with AZT. The effect of AZT on TE‑11 cell cycle distribution was determined by flow cytometry. Cellular DNA damage was evaluated by a comet assay and an MTT assay demonstrated that AZT significantly inhibited the viability of TE‑11 cells, in a time‑and dose‑dependent manner. In addition, TE‑11 cells treated with various concentrations of AZT exhibited a significant reduction in telomerase activity and percentage of cells in the G1/G0 phase, and an increase in the percentage of cells in the S phase. High doses of AZT caused DNA damage, and enhanced the expression levels of γ‑H2A histone family member X and phosphorylated checkpoint kinase 2 in TE‑11 cells. These results demonstrated that AZT effectively inhibits proliferation of the TE‑11 human esophageal cancer cell line in vitro. The growth inhibitory effects were associated with a reduction in telomerase activity, S and G2/M phase cell cycle arrest, and enhanced DNA damage, suggesting that AZT may be utilized in the clinic for the treatment of esophageal cancer.