Cancer stem cells (CSCs) underpin the resistance of breast cancer (BC) cells to therapy. Dendritic cell (DC)-based treatment is efficacious and safe, but the efficiency of this technique for targeting CSCs in BC treatment requires further investigation. The present study aimed to investigate the ability of DCs pulsed with breast CSC antigens to activate effector lymphocytes for killing BC cells. CD44/CD24 CSCs were isolated from BCA55-121, an in-house patient-derived BC cell line, and acquisition of stemness properties was confirmed by upregulated expression of OCT4A and a superior proliferative capacity in colony formation assays compared with whole population of BCA55-121 (BCA55-121-WP). DCs were differentiated from monocytes from peripheral blood of healthy donors and pulsed with CSC total RNA. Maturation of the CSC RNA-pulsed DCs was confirmed by increased expression of CD11c, CD40, CD83, CD86 and HLA-DR, as well as reduced CD14 expression compared with monocytes. Total lymphocytes co-cultured with CSC RNA-pulsed DCs were analyzed by flow cytometry for markers including CD3, CD4, CD8, CD16 and CD56. The results revealed that the co-cultures contained mostly cytotoxic CD8+ T lymphocytes followed by CD4+ T lymphocytes and smaller populations of natural killer (NK) and NKT cells. ELISA was used to measure IFN-γ production, and it was revealed that activated CD4 and CD8+ lymphocytes produced more IFN-γ compared with naïve T cells, suggesting that CD8+ T cells were effector T cells. CSC RNA was a more efficient antigen source compared with RNA from mixed BC cells for activating tumor antigen-specific killing by T cells. These CSC-specific effector T cells significantly induced BC cell apoptosis at a 20:1 effector T cell:tumor cell ratio. Of note, the breast CSCs cultures demonstrated resistance to effector T cell killing, which was in part due to increased expression of programmed death ligand 1 in the CSC population. The present study highlights the potential use of CSC RNA for priming DCs in modulating an anticancer immune response against BC.