Cancer stem cell (CSC) formation and epithelial-mesenchymal transition (EMT) are pivotal events in tumor cell invasion and metastasis. They have been shown to occur in resistance to tamoxifen. Moreover, microRNAs (miRNAs) have been associated with CSCs, EMT as well as tamoxifen resistance. Studying molecular mechanism of CSCs, EMT as well as tamoxifen resistance will help us to further understand the pathogenesis and progression of the disease and offer new targets for effective therapies. In the present study, we showed that miR-375 inhibits CSC traits in breast cancer MCF-7 cells. Bioinformatics analysis and experimental validation identified HOXB3 as a direct target of miR-375. Overexpressing miR-375 degraded HOXB3 mRNA in MCF-7 cells. Moreover, overexpression of HOXB3 induced formation of CSC phenotypes, EMT and tamoxifen-resistance as well as enhanced ability of migration and invasion in MCF-7 cells. Most ER-positive breast cancer-related deaths occur, because of resistance to standard therapies and metastasis, restoring miR-375 or targeting HOXB3 might serve as potential therapeutic approaches for the treatment of tamoxifen-resistant breast cancer.