Growth and production of type-B trichothecenes mycotoxins by the Fusarium graminearum strain CBS 185.32 were compared in GYEP medium supplemented with Mg2+ at different concentrations (0–4 mM). Mg2+ led to a strong decrease in toxin accumulation without affecting the mycelial growth, suggesting a specific Mg2+ effect on fungal secondary metabolism. Expression of Tri5, Tri6, and Tri12 genes was followed throughout the time courses of type-B trichothecenes (TCTB) yield in standard and 2 mM Mg2+-supplemented GYEP media. Mg2+ addition significantly decreased Tri5, Tri6, and Tri12 expression. The inhibition of toxin production by Mg2+ was shown to be highly correlated with inhibition of Tri5 transcription and, to a lesser extend, of Tri6 and Tri12. This is the first report of a transcriptional control of TCTB production by Mg2+.