Cancer immunotherapy is a promising intervention to fight against this global health problem. In particular targeting immune checkpoints, such as cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and programmed-death protein 1 (PD-1), by specific monoclonal antibodies is a current treatment for many malignances. A possible innovation in this field is based on the induction of humoral responses in the host by suppressing the effects of such immune checkpoints and as consequence favoring the activation of cellular immunity against the tumor cells. In this study, chimeric protein comprising the B subunit of Escherichia coli heat-labile enterotoxin as carrier and the extracellular domain of CTLA-4 (LTB-CTLA4) was produced in Nicotiana benthamiana by transient expression. The recombinant protein was accumulated up to 1.29 μg/g of leaves fresh weight on 4 day-post-infiltration. The integrity of the plant-made LTB-CTLA4 antigen was confirmed by western blot analysis and ELISA. Immunogenicity of the plant-made LTB-CTLA4 was assessed in BALB/c mice and the results showed that humoral responses were induced against both the LTB and CTLA-4 moieties. The plant-made LTB-CTLA4 stands as a promising candidate for the design of advanced protection studies against cancer in murine models.