Mixed lineage leukemias (MLLs) are histone-methylating enzymes with critical roles in gene expression, epigenetics and cancer. Although MLLs are important gene regulators little is known about their own regulation. Herein, to understand the effects of toxic stress on MLL gene regulation, we treated human cells with a common food contaminant mycotoxin, deoxynivalenol (DON). Our results demonstrate that MLLs and Hox genes are overexpressed upon exposure to DON. Studies using specific inhibitors demonstrated that Src kinase families are involved in upstream events in DON-mediated upregulation of MLL1. Sequence analysis demonstrated that the MLL1 promoter contains multiple Sp1-binding sites and importantly, the binding of Sp1 is enriched in the MLL1 promoter upon exposure to DON. Moreover, antisense-mediated knockdown of Sp1 diminished DON-induced MLL1 upregulation. These results demonstrated that MLL1 gene expression is sensitive to toxic stress and Sp1 plays crucial roles in the stress-induced upregulation of MLL1.