Despite the great improvement of patient outcomes by trastuzumab, a monoclonal antibody targeted on HER2-positive breast cancer, approximately 23% of patients with early-stage disease treated with adjuvant trastuzumab either fail to respond or experience recurrence within 10 years, highlighting the importance of identifying which HER2-positive patients would benefit from trastuzumab upfront. Efforts to identify biomarkers predictive of response to trastuzumab in initial breast tumor core biopsies have been complicated by the clinical and biological heterogeneity of HER2-positive tumors. Therefore, we identified a trastuzumab-resistant (TrR) signature that accurately predicts response to trastuzumab quantitively and qualitatively in vitro and in vivo, via repurposing transcriptome profiles in an engineered cell line model. We additionally demonstrated that our TrR signature was associated with tumor progression and capable of stratifying patient prognosis. Our study further illustrated the possible mechanism of this resistance as being less inherited cytotoxic T cell infiltration and failure to secrete Interferon-γ upon trastuzumab treatment in TrR tumors. These findings highlight the potential clinical application of TrR signature in treatment management and identifying possible immunotherapy interventions.