Abstract: Objective: To study the improving effect of Eupatilin (Eptl) on ethanol withdrawal (EtOHWI)-induced anxiety-like behavior in rats and probe the mechanisms related to ventral hippocampus (vHippo). Methods: Thirty-two adult male Sprague-Dawley rats were randomly divided into four groups, 8 rats per group: Saline-treated control group, EtOHWI model group, low-dose Eptl treatment group and high-dose Eptl treatment group. The EtOHWI was established by intraperitoneal injection of 3 g/kg of ethanol (20% volume/volume, dissolved in saline) once a day for 28 days followed by 3 days of withdrawal, during the withdrawal period, the low-and the high-dose Eptl treatment groups were respectively given 10 and 30 mg/kg Eptl through oral route once a day, while the saline control group was administered with an equal volume of saline. Thirty minutes after the third Eptl, all the rats were subjected to open filed (OF) and elevated plus maze (EPM) tests to detect anxiety-like behaviors. The serum coritosterone (CORT) concentration and vHippo γ-aminobutyric acid (GABA) secretion were measured by enzyme linked immunosorbent assay (ELISA), and vHippo glutamic acid decarboxylase 67 (GAD 67) mRNA relative expression was assayed by real-time quantitative polymerase chain reaction. The protein expression of GABAa receptor α1 (GABAaRα1), GABAaRα2, nuclear factor E2-related factor 2 (Nrf2), heme oxygense-1 (HO-1) in the vHippo were analyzed by Western blot. The levels of MDA, T-SOD, CAT and GSH, IL-6 and TNF-α were measured by commercial kits. Meanwhile, in the in vitro experiment, the nuclear levels of Nrf2 in HT22 cells were detected via immunofluorescent technique. Results: Compared with the rats in the EtOHWI group, the rats in low and high-dose Eptl treatment groups moving distance increased significantly (P<0.01) in the central region of OF which was 70.62% and 124.21% respectively, and moving time increased significantly (P<0.05 or P<0.01) which was 251.75% and 371.62% respectively in the central zone of the OF. Visited more frequently (P<0.05 or P<0.01) which was 110.33% and 207.32% respectively, and stayed time increased significantly (P<0.05 or P<0.01) which was 99.56% and 184.18% respectively in the open arms of the EPM1. In biochemical assays, compared with those in EtOHWI rats, in the rats of low-dose and high-dose Eptl treatment groups, the serum CORT concentrations decreased significantly (P<0.01). The vHippo GABA and GAD67 mRNA levels increased significantly (P<0.05 or P<0.01). The protein expression of GABAaRα1, GABAaRα2, Nrf2, HO-1 in the vHippo increased significantly (P<0.05 or P<0.01). The level of MDA decreased significantly (P<0.01), while the activities of T-SOD and CAT, as well as the level of GSH increased respectively significantly (P<0.05 or P<0.01). The levels of IL-6 and TNF-α decreased significantly (P<0.05 or P<0.01). In the in vitro experiment, the immunofluorescent assay showed that compared with blank control group, Nrf2 level in nucleus of HT22 stimulated by 200 μmol/L H₂O₂ increased significantly (P<0.01), whereas pretreatment with 30 μmol/L Eptl inhibited the increase of Nrf2 level (P<0.05). Conclusion: Eptl attenuates EtOHWI-induced anxiety-like behavior in rats, which may be mediated by regulating the vHippo GABAaR transmissional disorder of EtOHWI rats via its antioxidant and anti-inflammatory activities.