Prenylated flavonoids have been demonstrated to possess diverse bioactivities including antitumor effects. One new, daphnegiravone D ( 1), and four known ( 2– 5) prenylated flavonoids were isolated from Daphne giraldii. Their cytotoxic activities revealed that daphnegiravone D markedly inhibited the proliferation of cancer cells, but had no apparent cytotoxicity on human normal cells. Mechanistically, daphnegiravone D induced G0/G1 arrest and apoptosis, reduced the expression of cyclin E1, CDK2 and CDK4, and promoted the cleavage of caspase 3 and PARP in Hep3B and HepG2 cells. Meanwhile, daphnegiravone D increased the level of phosphorylated p38 and attenuated phosphorylated JNK. Further studies indicated that SB203580 partially reversed daphnegiravone D-induced G0/G1 arrest and apoptosis. The addition of SP600125 to both cell lines increased the cleavage of caspase 3 and PARP, but did not affect the G0/G1 arrest. Besides, in vivo studies demonstrated that daphnegiravone D obviously inhibited tumor growth in a nude mouse xenograft model through suppressing the proliferation of tumor cells, without significant effect on body weight or pathology characteristics. Taken together, the new compound selectively inhibited the proliferation of hepatoma cells via p38 and JNK MAPK pathways, suggesting its potential as a novel natural anti-hepatocellular carcinoma agent.