Abstract: Objective: Single factor experiment and response surface methodology were used to optimize the ultrasonic extraction of oridonin from Rabdosiaru bescens and the antitumor effect in vitro was studied. Methods: Three key factors including solid-liquid ratio, ultrasonic time, ultrasonic power were selected by one-factor-at-a-time experiments. HepG2 cells were cultured in vitro. The antitumor activity of oridonin was investigated by studying the survival rate of HepG2 cells and the expression of apoptotic proteins caspase-3 and caspase-9. Results: The optimum conditions of ultrasonic extraction of oridonin from Rabdosiaru bescens were as follows: Solid-liquid ratio was 1:18 g/mL, ultrasonic time was 1.5 h at 190 W ultrasonic power, the extraction yield of oridonin reached 4.122%±0.102%. In vitro, oridonin could significantly reduced the survival rate of HepG2 cells and increased the expression of caspase-3 and caspase-9. And when the concentration of oridonin exceeds 8 μmol/L, the survival rate and apoptotic protein of hepatoma cells were significantly affected (P<0.05). Conclusion: The maximum extraction yield of oridonin reached 4.122% by response surface methodology of ultrasonic extraction. In vitro pharmacological experiments confirmed that oridonin promoted cell apoptosis and reduced the survival rate of tumor cells through mitochondrial mediated endogenous pathway, and achieved the purpose of inhibiting tumor cells.