Chemotherapy remains the primary treatment modality for breast cancer (BCa) patients. However, chemoresistance commonly arises in clinical settings, contributing to poor prognosis. The development of chemoresistance is a dynamic and complex process involving the activation of oncogenes and inactivation of tumor suppressor genes. In this work, we utilized the RNA-sequencing (RNA-seq) technology to analyze the gene expression profiles of primary and recurrent tumor samples from BCa patients received the postoperative standard chemotherapy with doxorubicin (DOX), and identified glutathione S-transferase P1 (GSTP1) as a key factor in regulating chemoresistance. Molecular mechanistic studies revealed that high GSTP1 expression could not only impair the cytotoxicity of DOX by catalyzing the conjugation of reductive glutathione (GSH) with DOX, but also block the c-Jun NH2-terminal kinase (JNK) pathway to promote the proliferation via up-regulating anti-apoptotic B-cell lymphoma-2 (Bcl-2) expression. Given the severe side effects of DOX and the potential of RNA interference (RNAi) technology to silence target gene expression, we developed an endosomal pH-responsive nanoparticle (NP) platform for systemic co-delivery of DOX and GSTP1 siRNA (siGSTP1), and demonstrated its efficacy in reversing chemoresistance and suppressing the growth of DOX-resistant BCa tumors.