Specific inhibition on CDK9 has been proven to be a promising targeted cancer therapy. In this work, fourteen novel 2,4-disubstituted pyrimidine derivatives were designed and synthesized as potent and selective CDK9 inhibitors. These compounds showed broad anti-proliferative activities in various tumor cell lines, especially for PANC-1 cells with IC<sub>50</sub> values as low as 0.08 μM. The most selective compound 8d was 84-fold selective for CDK9 over CDK2. Mechanism study indicated that 8d induced apoptosis of PANC-1 cells and arrested the cell cycle at G2/M phase in a dose-dependent manner. Decreased phosphorylation of the CTD of RNAPII at Ser-2 and downregulation of CDK9 were confirmed in PANC-1 cells. Besides, Molecular docking was also performed to gain insights into the ligand-binding interactions of 8d inside CDK9 and CDK2 binding sites. In vivo studies indicated that 8d exhibited potent anti-tumor effects in PANC-1 xenograft models without causing obvious loss of body weight. Our research suggests that compound 8d, as a potent CDK9 inhibitor, can be considered as a good lead-candidate for further development.