Abstract: Objective To investigate the effect of 20（s）-Ginsenoside Rg3 （Rg3） on regulating autophagymediated sorafenib sensitivity of hepatocellular carcinoma cells. Methods Hep3B cells were used in this study and treated by Rg3，autophagy inhibitor 3-Methyladenine（3-MA），sorafenib，Rg3 plus sorafenib and 3-MA plus sorafenib. The sensitivity of Hep3B cells to Rg3，sorafenib and 3MA was detected by MTT method. Hep3B cells proliferation inhibition of Rg3，3-MA plus sorafenib were detected by CCK-8 method. The interaction of sorafenib with 3-MA and Rg3 was assessed by q value. LC3 conversion and LC3 turnover assay were used to detect the autophagic regulation effect of sorafenib and Rg3. Autophagy related proteins，including Bcelin1，LC3 and p62，were detected by Western blotting. Results MTT showed that Rg3，sorafenib and 3-MA inhibited Hep3B cell proliferation in a dose and time-dependent manner. CCK-8 test showed that the inhibition effects of low（0.5 μg／ml）and medium（1 μg／ml） doses of sorafenib in combination with Rg3 or with 3-MA were significantly higher than sorafenib alone with synergistic effect（P＜0.01）. High dose（2 μg／ml） of sorafenib in combination with Rg3 showed a higher inhibitory rate to Hep3B cells（P＜0.01），but no difference was observed on the inhibitory rate of high dose of sorafenib with or without 3-MA（P＞0.05）. The interaction of high dose of sorafenib in combination with Rg3 or 3-MA appeared addictive effect. LC3 conversion assay showed that with the extension of the duration of drug action，Rg3 and sorafenib increased the LC3-II level gradually. LC3 turnover assay showed that LC3 II level was significantly higher in sorafenib combined with CQ than that of sorafenib alone（P＜0.01），while Rg3 combined with or without CQ had no obvious changes of LC3-II levels（P＞0.05）. Western blotting showed that compared with control group， Rg3 and sorafenib increased Beclin1 protein expression obviously（P＜0.01）. Compared with sorafenib，Beclin1 decreased significantly when sorafenib combined with Rg3 instead of in combination with 3-MA. Rg3，3-MA and sorafenib caused the rising levels of LC3-II（P＜0.05）.LC3-II level in Rg3 combined with sorafenib was higher than that of sorafenib alone（P＜0.01），but there was no significant difference between 3-MA and 3-MA in combination with sorafenib（P＞0.05）. The level of p62 in Hep3B cells treated by 3-MA or sorafenib alone was significantly lower than that of control group（P＜0.05）.Rg3 combined with sorafenib increased p62 level significantly（P＜0.01），while sorafenib combined with 3-MA decreased p62 level significantly（P＜0.01）. Conclusion Sorafenib induced autophagy in hepatocellular carcinoma cells leads to decreased drug sensitivity by increasing the expression of Beclin1. Rg3 can increase the sensitivity of hepatocellular carcinoma to sorafenib possibly through inhibiting degradation of sorafenib-induced autophagy.