The intracellular superoxide level is a clue to clarification of the regulatory mechanism for mycotoxin production in Fusarium graminearum. In this study, we focused on two manganese superoxide dismutases (SODs) of the fungus, FgSOD2 and FgSOD3, to investigate the relationship of the superoxide level to trichothecene production. Recombinant FgSOD2 and FgSOD3 showed SOD activity, and they were localized mainly in the mitochondria and cytoplasm, respectively. Trichothecene production and mRNA levels of Tri5 and Tri6, which encode a trichothecene biosynthetic enzyme and a key regulator of trichothecene production, respectively, were greatly reduced in gene-deletion mutants of FgSod2 and FgSod3 (ΔFgSod2 and ΔFgSod3). Significant increases in the cytosolic and mitochondrial superoxide levels were observed in ΔFgSod2 and ΔFgSod3, respectively. These results suggested that the cellular superoxide level affects trichothecene production in F. graminearum.