HER2-positive breast cancer (HER2+ BC) is distinguished by its poor prognosis, propensity for early onset, and high risk of recurrence and metastasis. Consequently, anti-HER2-targeted therapy has emerged as a principal strategy in the treatment of this form of breast cancer. Pyrotinib, a novel irreversible pan-HER2 tyrosine kinase inhibitor, has brought fresh hope to patients with advanced HER2+ breast cancer. In this study, we conducted a comprehensive exploration of pyrotinib's antitumor mechanism. The in vitro results showed that pyrotinib significantly inhibited SKBR3 cells viability and induced apoptosis by promoting HER2 endocytosis and ubiquitylation, leading to HER2 degradation through the displacement of HSP90 from HER2. Beyond targeting the HER2 signaling pathway, pyrotinib also induced DNA damage, which was mediated by the activation of the reactive oxygen species/heat shock factor 1 signaling pathway and the downregulation of proliferating cell nuclear antigen expression. Furthermore, the in vivo results demonstrated a pronounced anticancer effect of pyrotinib in the SKBR3 xenograft mouse model, concomitant with a reduction in HER2 expression. In summary, our findings provide novel insights into the mechanism of pyrotinib in the treatment of HER2+ BC.