Histone H3 lysine79 (H3K79) methyltransferase DOT1L plays an important role in the activation and maintenance of gene transcription. It is essential for embryonic development as well as normal functions of the hematopoietic system, heart, and kidney in adults. DOT1L has been found to be a drug target for acute leukemia with mixed lineage leukemia (MLL) gene translocations. The rearranged onco-MLL can recruit DOT1L, causing aberrant H3K79 methylation, overexpression of leukemia relevant genes, and eventually leukemogenesis. Potent DOT1L inhibitors possess selective activity against this type of leukemia in cell-based and animal studies, with the most advanced compound being in clinical trials. In the medicinal chemistry point of view, we review the biochemistry, cancer biology, and current inhibitors of DOT1L, as well as biophysical (including X-ray crystallographic) investigation of DOT1L-inhibitor interactions. Potential future directions in the context of drug discovery and development targeting DOT1L are discussed.