Background <p>Human epidermal growth factor receptor 3 (HER3), a member of the HER/ErbB family, is broadly expressed across major breast cancer subtypes and contributes to tumor initiation, progression, and therapy resistance. Although numerous HER3-directed therapies have been evaluated in preclinical and clinical settings, limited efficacy and safety concerns have prevented any from achieving clinical approval. Near-infrared photoimmunotherapy (NIR-PIT) is a modality that selectively kills antibody-bound target cells upon NIR irradiation. In this study, we evaluated HER3-targeted NIR-PIT in breast cancer cell lines in vitro and in xenograft mouse models in vivo.</p> Methods <p>Triple-negative (MDA-MB-468) and Luminal (MCF-7) breast cancer cells were utilized. In vitro, the therapeutic efficacy of HER3-targeted NIR-PIT was assessed by monitoring morphological changes via differential interference contrast microscopy and evaluating cell cytotoxicity using propidium iodide staining. In vivo, MDA-MB-468 and MCF-7 were subcutaneously xenografted into nude mice. Mice bearing MDA-MB-468 tumors were assigned to Control, HER3 antibody-photoabsorber conjugate i.v., and HER3-targeted NIR-PIT groups, while MCF-7 tumor-bearing mice were divided into Control and HER3-targeted NIR-PIT groups. Tumor growth and survival rates were monitored, and histological alterations post-NIR-PIT were analyzed using Hematoxylin and eosin (H&amp;E) and pan-cytokeratin immunohistochemical staining.</p> Results <p>HER3-targeted NIR-PIT induced robust cancer cell death in vitro, significantly suppressed tumor growth and prolonged survival in vivo, and achieved complete responses in 30% of treated mice bearing triple-negative breast cancer xenografts.</p> Conclusions <p>These findings support HER3-targeted NIR-PIT as a promising therapeutic strategy for HER3-positive breast cancer.</p>