Gastric cancer (GC) as the third most common cause of cancer-associated mortality worldwide is one of the cancers with very high heterogeneity. Cancer stem cells (CSCs) as a small subset of cancer cells in solid tumors with the self-renewal, differentiation and tumorigenic ability are responsible for tumor initiation, progression, recurrence, metastasis, and resistance to current treatments. Therefore, eradication of CSCs is very vital to cure cancer. Here, we first isolated and identified sphere-forming cells in tumor tissue from four GC patients and then analyzed T cell responses induced by monocyte-derived dendritic cells (DCs) loaded with total mRNA of sphere-forming cells in terms of interferon-gamma (IFN-γ) gene expression and specific cytotoxicity. Spheroid colonies were formed in serum-free media. Sphere-forming cells dissociated from tumorspheres heterogeneously expressed CD44, CD54, and epithelial cell adhesion molecule (EpCAM) markers and generated one tumor in nude mice. These results demonstrated that gastric CSCs were enriched in tumorspheres. Cytokine-matured DCs loaded with mRNA of sphere-forming cells were able to induce IFN-γ gene expression in T-lymphocytes after a 12-day co-culture. mRNA level of IFN-γ gene in these lymphocytes was more highly expressed compared to stimulated T-lymphocytes by DCs transfected with normal tissue (6.4-9.39 folds). Cytotoxic activity of primed T-lymphocytes with antigens of sphere-forming cells was significantly higher than normal tissue antigens and mock DCs (P ≤ 0.0001). Taken together, DCs loaded with mRNA of sphere-forming cells that elicit effectively specific T cell-mediated immune responses in vitro, may be considered as a promising therapeutic vaccination in GC patients in future.