Esophageal squamous cell carcinoma (ESCC) is a tumor composed of heterogeneous cells that easily become radioresistant, which leads to tumor recurrence. The most commonly used treatment for ESCC is fractionated irradiation (FIR) therapy that utilizes ionizing radiation to directly induce cytotoxic cell death. However, this treatment may not be able to eliminate all cancer cells due to high adaptive evolution. To determine whether the transcriptome dynamics during ESCC recurrence formation are associated with FIR response, an in vitro cell culture model for ESCC radioresistance that mimics the common radiotherapy process in patients with ESCC was established in the present study. High-throughput sequencing analysis of in vitro cultured ESCC cells was performed using different cumulative irradiation doses, as well as tumor samples from FIR-treated patients with ESCC before and after the development of radioresistance. Radioresistance-associated genes and signaling pathways that were aberrantly expressed in radioresistant ESCC cells were identified, including autophagy-related 9B (regulation of autophagy), DNA damage-inducible transcript 4, myoglobin and plasminogen activator tissue type, which are associated with response to hypoxia, Bcl2-binding component 3, tumor protein P63 and interferon gamma-inducible protein 16, which are associated with DNA damage response. The heterogeneity and dynamic gene expression of ESCC cells during acquired radioresistance were further studied in primary (41 single cells), 12 Gy FIR-treated (87 single cells) and 30 Gy FIR-treated (89 single cells) cancer cells using a single-cell RNA sequencing approach. The results of the present study comprehensively characterized the transcriptome dynamics during acquired radioresistance in an in vitro model of ESCC and patient tumor samples at the population and single cell level. Single-cell RNA sequencing revealed the heterogeneity of irradiated ESCC cells and an increase in the radioresistant ESCC cell subpopulation during acquired radioresistance. Overall, these results are of potential clinical relevance as they identify a number of signaling molecules associated with radioresistance, as well as opportunities for the development of novel therapeutic options for the treatment of ESCC.