Background: Radiotherapy has an unsatisfactory effect on gastric cancer. The purpose of this study was to investigate the effect of pyrotinib-a highly effective human epidermal growth factor receptor (HER) family inhibitor, on the radiosensitivity of HER2-positive gastric cancer and its mechanism in vivo and in vitro. Methods: NCI-N87 and SNU-216 were HER2-positive gastric cancer cell lines, treatment with or without 0.01 mu M pyrotinib 12 hours before irradiation, the proliferation capacity was determined by CCK8, and clone formation experiments were used to test the radiosensitization effect of pyrotinib. The expression of HER2, gamma-H2AX, apoptosis protein, senescence-associated proteins, and AKT/ERK signaling pathway changes were determined by Western blot. Cell cycle and apoptosis were established by flow cytology. Immunofluorescence was utilized to detect the expression of HER2 and gamma-H2AX. Senescence cells were stained by beta-galactosidase staining method. Pathway enrichment analysis was tested by RNA sequencing. Next, a xenograft tumor model was constructed in nude mice for verification in vivo. Results: The clone formation experiment indicated the radiosensitivity of pyrotinib. The combined treatment can inhibit the entry of HER2 protein into the nucleus and reduce the phosphorylation of the ERK signaling pathway caused by irradiation. Pyrotinib also enhancing DNA damage, inducing apoptosis, G2/M phase arrest, and promoting senescence. In the xenograft model, the tumor inhibition rate was significantly stronger in the combined treatment group. Conclusions: Our results demonstrated that pyrotinib can induce radiosensitivity in HER2-positive gastric cancer in vivo and in vitro. This effect is through reducing irradiation-induced HER2 entry into the nucleus and inhibiting ERK1/2 signaling pathway.