Long-term regular ethanol intake could change the gut microbiota and affect anxiety and depression-like behaviors․ It is not clear that whether ethanol withdrawal after short-term low-dose drinking has an effect on the gut microbiota or whether it is related to anxiety-like behaviors․ In this study, 30 male Sprague￣Dawley rats were randomly divided into three groups: Ethanol-C: ethanol treatment group, treated with (5 g / kg, 25% V / V) ethanol for 14 days; Ethanol-2: ethanol withdrawal group, treated with (5 g / kg, 25% V / V) ethanol for 14 days and then withdrawal for one day; Ethanol-0: Control group, the rats were given the same amount of distilled water for 14 days․ Feces were collected from all rats, and high-throughput sequencing methods were used to analyze the effect of ethanol withdrawal after short-term low-dose drinking on the gut microbiota․ The open-field test and elevated plus-maze test were used to determine anxiety-like behaviors, and analyze the correlation between gut microbes and anxiety-like behaviors caused by alcohol withdrawal․ Taxonomic analysis of gut microbiota found that the composition and abundance in the ethanol withdrawal group were significantly different from those in the control group and the alcohol-treated group․ The Alpha diversity of gut microbiota in the ethanol withdrawal group was not significantly different from the control group and the ethanol treatment group, whereas the microbial community structure was significantly different․ The percentage of time spent in the open arms and total distance of rats in the ethanol withdrawal group were significantly reduced (P < 0․ 05) , and behavioral parameters were significantly positive correlated with Bacteroides, Fusobacterium, and Escherichia-Shigella (P < 0․ 05) , but significantly negative correlated with Rumenococcus, Trichospirillum and other bacteria (P < 0․ 05) ․ This study suggests that short-term low-dose ethanol withdrawal does not affect the Alpha diversity, but can change the abundance and community structure of the gut microbiota in rats, and the gut microbiota are correlated with anxiety-like behaviors in rats․ This study clarified the changes of gut microbiota after short-term low-dose ethanol withdrawal, and provided a new direction for the study of anxiety-like behaviors caused by short-term low-dose ethanol withdrawal․