Through a structure-guided rational drug design approach, we have discovered a highly selective inhibitor compound 40 (JSH-150), which exhibited an IC of 1 nM against CDK9 kinase in the biochemical assay and achieved around 300-10000-fold selectivity over other CDK kinase family members. In addition, it also displayed high selectivity over other 468 kinases/mutants (KINOMEscan S score(1) = 0.01). Compound 40 displayed potent antiproliferative effects against melanoma, neuroblastoma, hepatoma, colon cancer, lung cancer as well as leukemia cell lines. It could dose-dependently inhibit the phosphorylation of RNA Pol II, suppress the expression of MCL-1 and c-Myc, arrest the cell cycle and induce the apoptosis in the leukemia cells. In the MV4-11 cell-inoculated xenograft mouse model, 10 mg/kg dosage of 40 could almost completely suppress the tumor progression. The high selectivity and good in vivo PK/PD profile suggested that 40 would be a good pharmacological tool to study CDK9-mediated physiology and pathology as well as a potential drug candidate for leukemia and other cancers.