HER2-positive breast cancer is characterized by high proliferative and metastatic potential, and Herceptin (Trastuzumab) is commonly used as a targeted therapy. However, approximately one-third of patients eventually develop drug resistance, leading to reduced therapeutic efficacy. In this study, RNA sequencing was performed to compare gene expression profiles between wild-type and Herceptin-resistant HER2-positive breast cancer cells, revealing a significant upregulation of the ALOX5 gene in resistant cells. Subsequent validation using RT-PCR, Western blotting, and immunohistochemistry (IHC) confirmed that both ALOX5 mRNA and protein levels were markedly higher in resistant cells and tumor tissues compared with the wild-type group. Analysis of TCGA clinical data further indicated that elevated ALOX5 expression strongly correlates with poor prognosis and increased metastasis in patients. Functionally, treatment with an ALOX5 inhibitor effectively reduced the viability of resistant cells, and its combination with Herceptin produced synergistic inhibitory effects. Moreover, CRISPR/Cas9-mediated knockout of ALOX5 significantly suppressed cell proliferation. Collectively, these findings suggest that ALOX5 plays a crucial role in the development of Herceptin resistance, and its inhibition may enhance therapeutic response. This study identifies ALOX5 as a potential molecular target for overcoming resistance in HER2-positive breast cancer.