Protein methyltransferases (PMTs) are involved in numerous biological processes and have been studied as a promising target class in the field of oncology and other diseases. Disruptor of telomeric silencing 1-like (DOT1L), a histone H3 lysine 79 (H3K79) methyltransferase, plays an important role in the progressions of mixed-lineage leukemia (MLL)-rearranged leukemias and has been validated as a potential therapeutic target. Here we report the discovery and characterization of a novel DOT1L inhibitor, massonianoside B (MA), by pharmacophore-based in silico screening and biological studies. MA is a structurally unique natural product inhibitor of DOT1L with an IC50 value of 399 nM. The compound displays high selectivity for DOT1L over other S-adenosylmethionine (SAM)-dependent PMTs. Treatment of MLL-rearranged leukemia cells with MA gives a dose-dependent reduction in cellular levels of histone lysine 79 mono- and dimethylation without affecting the methylation of other histone sites. Moreover, MA selectively inhibits proliferation and causes apoptosis in MLL-rearranged leukemia cells and downregulates the expression of MLL fusion target genes, including HOXA9 and MEIS1. Molecular docking analysis revealed that MA may bind to the SAM-binding site of DOT1L. We identified MA as not only a novel DOT1L inhibitor with antileukemic activity but also a DOT1L-targeted molecular probe that may serve as a useful chemical tool for investigating the role of DOT1L in biological processes.