Human epidermal growth factor receptor2 (HER2) overexpression/amplification is associated with high malignancy, rapid disease progression and poor overall survival in breast cancer. The application of anti-HER2 drugs has greatly improved the survival of patients with HER2-positive breast cancer, but drug resistance issues affect the long-term efficacy. The HER2 mutation is considered to be one of the reasons for resistance to antiHER2 therapy, and there is currently no standard treatment. We report for the first time the detection of HER2 amplification with R157W mutation by second-generation sequencing (NGS) in a 57-year-old hormone receptor-negative, HER2-positive woman with advanced breast cancer who was resistant to multi-line anti-HER2 therapies. She subsequently received pyrotinib combined with capecitabine treatment and achieved partial response. The smallmolecule pan-HER family irreversible inhibitor pyrotinib combined with capecitabine has shown a promising effect in the treatment of HER2 mutation-induced resistance, but the molecular mechanism and efficacy need to be further verified.