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Background Neoadjuvant therapy with trastuzumab, pertuzumab and paclitaxel (THP) has significantly improved the prognosis of patients with human epidermal growth factor receptor 2 (HER2)+ breast cancer (BC). However, there remains a subset of non-responsive patients. Thus, this study sought to identify key regulators of THP neoadjuvant therapy resistance and potential targets to sensitize sensitivity.Methods The Cancer Genome Atlas database, Gene Expression Omnibus and membrane protein database were used to identify the key regulator of THP neoadjuvant resistance. The biological functions and mechanisms of delta-like 4 proteins (DLL4) in THP therapy resistance were investigated in vitro and in vivo using the bioinformatic analysis, multiplex immunofluorescence, flow cytometry, sphere formation assays and chromatin immunoprecipitation, etc. Furthermore, DLL4-targeted chimeric antigen receptor (CAR)-T cells were established to sensitize THP therapy.Results DLL4 was identified as a key target in THP neoadjuvant therapy resistance for HER2+ BC. Mechanistically, DLL4+ tumor cells exhibited enhanced stemness and resistance to the THP neoadjuvant chemotherapy. Additionally, soluble DLL4 can split away from tumor cells and diffuse into the stroma, where it can activate the Notch signaling pathway in neutrophils, inducing the formation and release of neutrophil extracellular traps (NETs) by regulating the transcription of MPO, PDIA4 and ELANE. This led to the exclusion of lymphocyte infiltration, thereby enhancing therapy resistance. What is more, we designed a DLL4-targeted CAR-T to eliminate DLL4+ tumor cells and reverse the resistant status.Conclusions Our study revealed novel functions of DLL4 in cell stemness and immune infiltration, including NET formation and T cell exclusion, which collectively contributed to THP neoadjuvant therapy resistance in HER2+ BC. Furthermore, we provided a CAR-T-based therapy to sensitize the THP neoadjuvant therapy.