Dendritic cell (DC) vaccination targeting cancer stem cells is an effective way to suppress tumor progression and reduce the metastasis and recurrence. In the present study, we explored the suitability of side population (SP) cells as source of antigens for DC vaccination against hepatocellular carcinoma (HCC) in a mouse model. In this study, we identified the „stem-like“ characteristics of SP cells in the MHCC97 and Hepa 1-6 HCC cell lines. We found that SP cells express high levels of tumor-associated antigens and MHC class I molecules. Although loading with cell lysates did not change the characteristics of DCs, SP cell lysate-pulsed DCs induced antigen-specific T cell responses, including T cell proliferation and increased IFN-γ production by stimulated CD8(+) T cells. We investigated the cytotoxicity of T cells stimulated by SP cell lysate-pulsed DCs in nude mice co-injected with MHCC97 cells. To mimic the in vivo environment, we also confirmed the result in mouse HCC cell line Hepa 1-6 induced tumor-bearing C57/BL6 immune competent mice, and we demonstrated that vaccination with DCs loaded with Hepa 1-6 SP cell lysates could induce a T cell response in vivo and suppress the tumor growth. Our results may have applications for anti-HCC immunotherapy by targeting the cancer stem cells and may provide new insight for cancer vaccines.