Dexamethasone (Dex), a synthetic glucocorticoid that acts by binding to the glucocorticoid receptor (GR), has been widely applied to treat leukemia and lymphoma, however the precise mechanism underlying Dex action is still not well elucidated. DOT1L, a histone H3-lysine79 (H3K79) methyltransferase, has been linked to multiple cancer types, particularly mixed lineage leukemia (MLL) gene rearranged leukemia, but its contribution to lymphoma is yet to be delineated. Analysis from TCGA database displayed that DOT1L was highly expressed in lymphoma and leukemia. In the present study, we initially demonstrated that DOT1L served as a newly target gene controlled by GR, and downregulation of DOT1L was critical for the killing of B lymphoma cells by Dex. Further study revealed that Dex had no impact on the transcriptional activity of DOT1L promoter, rather it reduced the mRNA level of DOT1L through decreasing mRNA stability. In addition, knockdown of DOT1L remarkably inhibited the B lymphoma cells growth. Overall, our findings indicated that DOT1L may serve as a potential drug target and a promising biomarker of Dex sensitivity when it comes to treating B lymphoma.