Abstract: Objective: To explore the effects of paeoniflorin on alcohol withdrawal induced anxiety-like behavior, inflammatory factors and intestinal microorganisms in a long-term low-dose drinking and then withdrawal rats model. Methods: 24 Sprague Dawley rats were randomly divided into 3 groups with 8 rats in each group. Except the control group (NCG), the rats were treated with alcohol (5 g/kg and 25% v/v) once a day by gavage for 28 days. Then 3 days of alcohol withdrawal . During the withdrawal period, the rats in the paeoniflorin treatment group (PAG) were gavaged with a dose of 50 mg/(kg·BW), and the model group (MCG) and control group (NCG) were gavaged with the same amount of distilled water. At the end of the experiment, behavioral test was carried out, and the rat serum was collected to determine the inflammatory factors interleukin-6 (IL-6), interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α）. Fecal samples were collected for high-throughput sequencing to analyze the changes in the microbiota. Results: In the elevated plus maze (EPM) and open field experiment (OF), the percentage of open arm time (PTO) and the total distance of spontaneous movement (TD) were the lowest in the MCG group. Paeoniflorin could significantly alleviate the anxiety-like behavior induced by alcohol withdrawal in rats (P<0.05), and paeoniflorin could significantly reduce the level of serum inflammatory factors IL-6, TNF-α and IL-1β. The correlation analysis showed that there was a significant positive correlation between anxiety-like behavior and inflammatory factors (P<0.05). 16S rDNA sequencing showed that paeoniflorin had no significant effect on the alpha diversity of gut microbiota in alcohol withdrawal rats (P>0.05), whereas the paeoniflorin could significantly change the β diversity and microbial community structure (P<0.05). Paeoniflorin could significantly increase the relative abundance of Ruminococcaccae UCG-005 and Holdemanella (P<0.05), and reduce relative abundance of Romboutsia and Candidatus saccharimonas. There was a significant negative correlation between Holdemanella and serum inflammatory factor IL-6 (P<0.05). Candidatus saccharimonas was significantly positively correlate with IL-6, IL- 1β and TNF-α (P<0.05). Conclusion: Paeoniflorin could effectively alleviate the anxiety-like behavior induced by alcohol withdrawal in rats, reduce the level of serum inflammatory factors, change the structure of intestinal microbial community, increase the relative abundance of anti-inflammatory related bacteria and reduce the relative abundance of pathogenic bacteria.