Epithelial membrane protein 2 (EMP2) plays crucial roles in cell proliferation, migration, and adhesion. Despite its importance, conventional EMP2 RNAi therapy shows limited efficacy in vivo. We therefore developed a novel RNA-delivery system utilizing self-assembling defense peptide-cholesterol conjugates for efficient EMP2-siRNA transfection. The engineered HH2-siEMP2 nanoparticles exhibited optimal size and positive surface charge, conferring excellent serum stability and enhanced cellular uptake in breast cancer cell lines. These nanoparticles effectively silenced EMP2 expression, leading to significant suppression of tumor migration and invasion in both in vitro and in vivo models. Beyond direct anti-tumor effects, the HH2-C conjugate demonstrated immunomodulatory properties by promoting Th1 cell expansion, reducing Th2 cells and immunosuppressive Tregs, and restoring Th17/Treg balance. These findings establish EMP2 as a promising therapeutic target in breast cancer and highlight the potential of HH2-C-based nanoparticles as a dual-function platform combining efficient siRNA delivery with immunostimulatory activity.© 2025 The Authors.