To illustrate the differences in toxicities between deoxynivalenol (DON) and nivalenol (NIV), cell proliferation, cytokine secretion, and the involvement of heat shock protein 90 (Hsp90) were investigated. Both toxins retarded the proliferation of all four cell lines tested. NIV was more potent than DON in human promyelocytic leukemia cell line HL60, human lymphoblastic leukemia cell line MOLT-4, and rat aortic myoblast cell line A-10. In contrast, both toxins exhibited almost equivalent potencies in human hepatoblastoma cell line HepG2. If both toxins exert their toxicities through the same mechanism, one should be more potent than the other, regardless of cell types. While exposure to DON significantly induced the secretion of anti-hematopoietic cytokines macrophage inflammatory protein-1α (MIP-1α/CCL3) and MIP-1β/CCL4, treatment with NIV decreased the secretion of these cytokines in HL60 cells, indicating that the toxicity mechanisms of these toxins differ. An Hsp90-specific inhibitor radicicol canceled the effect of DON on these cytokine secretions, indicating that Hsp90 plays a crucial role in these DON-induced cytokine secretions in HL60 cells. Conversely, the results of treatment with NIV and radicicol indicate that radicicol does not mitigate the effect of NIV. When viewing the above results collectively, although these toxins share highly similar chemical structures, there are evident differences in their toxicities.