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Background: Although activation of human epidermal growth factor receptor 3 (HER3) is linked to resistance to targeted therapies in several cancer types, the HER3 expression profile during pancreatic cancer treatment remains unknown. Aims: We evaluated the HER3 expression status after chemotherapy for pancreatic cancer and its association with clinicopathological features and clinical outcomes. Materials & Methods: We included patients with pancreatic cancer who underwent chemotherapy and whose post‐treatment archival tissue specimens were collected. HER3 expression was retrospectively assessed by immunohistochemistry scoring (0, 1+, 2+, and 3+) of the membranous staining intensity. Results: HER3 expression after chemotherapy was evaluated in 41 patients, with matched‐pair analysis in five patients before and after chemotherapy. HER3 expression was observed in most of the patients after chemotherapy, demonstrating IHC scores of ≥ 1+ and ≥ 2+ in 40 (98%) and 26 (63%) of 41 patients, respectively. Of the 38 patients with adenocarcinoma, the median overall survival in the HER3 (2+/3+) and HER3 (0/1+) groups was 21.0 and 17.1 months, respectively. The comparison of HER3 expression before and after chemotherapy performed in five cases revealed that scores changed from 2+/3+ to 0/1+ in one case, 0/1+ to 2+/3+ in another case, and remained at 2+/3+ in three cases. Cancer genome profiling tests in eight cases found no HER3 amplification or mutation, and seven of these cases had adenocarcinomas with KRAS and TP53 mutations. Conclusion: A high prevalence of HER3 expression was observed in pancreatic cancer patients after chemotherapy. Our findings indicate that HER3 is a potential therapeutic target for pancreatic cancer, deserving further clinical investigation. [ABSTRACT FROM AUTHOR]