Fumonisin B1 (FB1) is one of the most toxic mycotoxins and is harmful to humans and animals due to its hepatotoxicity, immunotoxicity and carcinogenicity. However, the mechanism of its immunosuppressive effect is still under investigation. Dendritic cells (DCs) are the most potent professional antigen-presenting cells, and their differentiation, maturation and immunomodulatory functions are closely related to the immunotoxicity of certain mycotoxins. Migratory capacity is a prerequisite for mature DCs (mDCs) to move and present antigens in secondary lymphoid tissue, whereas the mechanical properties and cytoskeletal structure are critical for their migration and immune functions. Therefore, the effects of FB1 on the cell viability, mechanical characteristics, cytoskeletal structure and its binding proteins, migration, co-stimulatory molecules and the immune functions of mDCs were investigated to explore the potential mechanisms of immunotoxicity. The results showed that FB1 could impair the chemotactic migratory capability, the expression of co-stimulatory molecules and the ability of DCs to stimulate T cell proliferation. Further analyses elucidated that the mechanical properties of mDCs were changed, the cytoskeletal structures were reorganized and the expressions of cytoskeleton-binding proteins were regulated. In conclusion, the attenuated migration and immune functions of mDCs caused by FB1 may be related to their altered mechanical properties and cytoskeleton remodeling, which may be one of the action modes for FB1 to exert its immunosuppressive effect.