Purpose: Trastuzumab, a monoclonal antibody, is widely employed for targeting human epidermal growth factor receptor 2 ( HER2 ) in patients with advanced breast cancer (BC). However, the application of trastuzumab is limited, since only 25%-30% of BC patients exhibit a high HER2 expression level. This study aimed to expand the therapeutic scope of trastuzumab in BC treatment. Methods: Gain-of-function studies were performed to evaluate the effects of various treatments on breast cancer cell proliferation, migration, invasion and apoptosis. Additionally, the cooperative effects of calcitriol and trastuzumab were investigated using xenograft models. Results: Dicer over- expression was observed to induce HER2 overexpression in triple-negative breast cancer (TNBC) MDA-MB-231 cells while simultaneously suppressing their proliferative, migratory, and invasive properties. Calcitriol-induced Dicer expression was found to drive TNBC cells into a HER2-overexpressed state, rendering them suitable for trastuzumab treatment. This induction also inhibited the proliferation, invasion, and migration of BC cells. Moreover, calcitriolmediated HER2 overexpression through the upregulation of Dicer expression enhanced trastuzumab's efficacy in suppressing TNBC cell proliferation and migration. In xenograft models, this combination accelerated trastuzumabinduced inhibition of TNBC tumor growth. Conclusion: The findings of this study demonstrate that calcitriol may expand the therapeutic applications of trastuzumab for TNBC patients by initiating HER2 overexpression. Consequently, Dicer may broaden the clinical application of HER2 antibody therapy for TNBC patients who were previously not candidates for anti-HER2 HER2 antibody treatment.