We studied the effect of dehydrocostus lactone (DHL) on the biological characteristics of HepG2 human hepatocellular carcinoma cells. The inhibition of cell viability by different concentrations of DHL (10, 20, 40, 80, and 160 mu mol/liter) was measured using MTT test. As the determined half-maximum inhibitory concentration (IC50) was 20.33 mu mol/liter, DHL in a concentration of 20 mu mol/liter was used in further experiments. Cell proliferation, migration, invasion ability, and apoptosis were assessed by Ki-67 immunofluorescence, Transwell assay, and TUNEL analysis. The level of p-AKT protein was determined by Western blotting. DHL significantly inhibited the viability, proliferation, migration, and invasion of HepG2 cells in comparison with the control group, and induced cells apoptosis. DHL down-regulated the expression of p-AKT protein in the HepG2 cells in comparison with the control group. PI3K/AKT signaling pathway activator 740Y-P could block the above-mentioned effects of DHL. Thus, DHL inhibits the malignancy of HepG2 human hepatocellular carcinoma cells via down-regulation of PI3K/AKT signaling pathway.