Endocrine therapy, using tamoxifen (TAM) and aromatase inhibitors is commonly used to treat estrogen receptor (ER)-positive breast cancer. Despite the remarkable benefits, resistance against TAM presents a serious therapeutic challenge. Although multiple mechanisms responsible for endocrine resistance have been suggested, the identification of new biomarkers is still strongly required. Since several HOX transcription factors have been proposed as strong candidates in the development of resistance against TAM therapy in breast cancer, we generated an in vitro model of acquired TAM resistance using ER-positive MCF7 breast cancer cells (MCF7-TAMR), and analyzed the expression pattern and the epigenetic states of HOX genes. Gain- and loss-of-function experiments were performed to investigate the functional significance of HOX gene expression on TAM sensitization and resistance, and then the impact of HOX gene expression on patient survival was examined using publicly available datasets. HOXB cluster genes were uniquely up-regulated in MCF7-TAMR cells. Survival analysis of in slico data showed the correlation of high expression of HOXB genes with a poor response to TAM in the case of ER-positive breast cancer patients treated with TAM therapy. The overexpression of multi HOXB genes in MCF7 renders cancer cells more resistant to TAM, whereas the knockdown restores TAM sensitivity. Furthermore, the activation of HOXB genes in MCF7-TAMR was associated with the regulation of histone modifications, particularly the gain of H3K9ac. These findings imply that the activation of HOXB genes are involved in the development of TAM resistance, and therefore, could serve a target for developing new strategies to prevent or reverse TAM resistance.