BACKGROUND Trastuzumab deruxtecan (T-DXd) has shown promising efficacy against human epidermal growth factor receptor 2 (HER2)-positive gastric and gastroesophageal junction (GEJ) adenocarcinomas. The efficacy of T-DXd rechallenge, however, has remained unclear. This is the first report of a dramatic response to T-DXd rechallenge in a patient with HER2-positive GEJ adenocarcinoma after confirmation of HER2 overexpression immediately prior to the rechallenge. CASE REPORT A 67-year-old man was diagnosed with HER2-positive gastric cardia (or GEJ) adenocarcinoma with lymph node and liver metastases. Initial T-DXd therapy was started as fourth-line chemotherapy. The best response was partial, and progression-free survival was 5.6 months. After an immune checkpoint inhibitor-based regimen, a rechallenge with T-DXd was planned as a seventh-line treatment. HER2 overexpression was confirmed by re-biopsy immediately before the rechallenge. He is currently receiving T-DXd without progression or severe treatment-related adverse events. CONCLUSIONS This is the first case report of a response to T-DXd rechallenge in a patient with HER2-positive gastric cancer. This rechallenge could be considered a treatment strategy for HER2-positive gastric cancer, for cases in which the initial T-DXd treatment was effective. Confirmation of HER2 overexpression and re-biopsy immediately before the rechallenge would be important for this strategy.