Radiation therapy is a widely used treatment for esophageal cancer. However, radiation resistance might result in a poor prognosis. Overexpression of HER2 has been related to adaptive radiation resistance. Pyrotinib is a HER2 inhibitor that shows an anti-tumor effect in breast cancer. The present study aims to explore the influence of pyrotinib combined with radiotherapy on HER2-positive esophageal cancer cells and explore the underlying mechanism. We screened two cell lines (TE-1 and KYSE30) that highly express HER2 from several human esophageal cancer cell lines. Cells were treated with pyrotinib or/and radiation. Cell proliferation, cell cycle distribution, and cell migration were measured. The protein levels involved in cell cycle and DNA repair were measured by Western blot. Results showed that pyrotinib inhibited HER2 activation and exerted an anti-proliferative effect in TE-1 and KYSE30 cells. Furthermore, it enhanced the anti-proliferative effect of radiation in these two cell lines. These effects might be via inhibiting HER2 phosphorylation, inducing G0/G1 arrest, and reducing EMT and DNA repair. Our results indicated that pyrotinib sensitivitied HER2 positive esophageal cancer cells to radiation treatment through various mechanisms. These findings may provide a new therapeutic strategy for treating HER2 positive esophageal cancer.