A series of enmein-type diterpenoid amino acid ester derivatives (14-22) were designed and synthesized according to l-alanine-(14-oridonin) ester trifluoroacetate (clinical candidate HAO472). Their antiproliferative activities were tested against SGC-7901, Bel-7402, HL-60, PC-3, A549 and K562 cancer cell lines and L-02 normal liver cells. The results showed that compound 19 possessed the most potent cytotoxicity with IC50 s at sub-micromolar level against human hepatoma Bel-7402 and chronic myelogenous leukemia K562 cells and more potent than l-alanine-(14-oridonin) ester (23). More importantly, 19 displayed 70-fold less cytotoxicity than parent 3 (IC50 = 25.47 μM) against L-02 cells, which exhibited certain selectivity. Further mechanism study in Bel-7402 cells revealed that 19 could induce apoptosis, G1 phase cell cycle arrest and mitochondrial dysfunction. Western blot results of caspase-3, Bax and cytochrome c upregulation and pro-caspase-3, Bcl-2 and Bcl-xL downregulation confirmed the intrinsic pathways. Overall, these data collectively demonstrated the high efficiency and selectivity of 19, l-phenylalanine-enmein-type diterpenoid ester, which inspires further and effective application as a potential antitumor candidate.