To explore the potential mechanism of Guizhi Fuling Pill（桂枝茯苓丸，GFW）in the treatment of adenomyosis（AM）by combining gut microbiota and metabolomics technology.

Seven-week-old ICR mice were randomized assigned to the control，model and GFW groups. The AM model was developed by the allogeneic pituitary transplantation method. The three groups were sacrificed after 8 weeks of intragastric administration. Histopathological changes of the mice uterus were evaluated，and the frequency and amplitude of uterine contraction in vitro were detected.16SrRNA gene sequencing was utilized to compare the changes in the Alpha and Beta diversity of the intestinal flora before and after GFW intervention，and to screen the differential gut microbiota. The changes of gut metabolites after GFW treatment were explored using liquid chromatography-mass spectrometry（LC-MS）. Correlation analysis was performed on the differential microbiota and metabolites to find out the possible mechanism of GFW regulating AM，followed by experimental validation.

HE staining confirmed that the AM model was successfully formed. Compared with those of the model group，the amplitude of uterine contraction in vitro was significantly decreased after GFW treatment（P<0.05），and the contraction frequency showed a downward trend. The Alpha diversity of gut microbiota showed that there was no significant difference in Observed species and Ace index among the three groups. The distribution of gut microbiota varied significantly between the control and model groups，according to the Beta diversity results. GFW treatment was able to modulate the gut flora composition of AM mice to varying degrees. Further analysis indicated that the abundance of Bacteroidetes and Bacteroides in the intestine of AM mice was significantly down-regulated（P<0.01，P<0.01），whereas the relative abundance of Firmicutes and Tenericutes was significantly up-regulated（P<0.05，P<0.05）. After GFW intervention，the abundance of Tenericutes，Mycoplasma and Enterorhabdus was significantly decreased（P<0.05，P<0.05，P<0.01），while Parabacteroides was significantly increased（P<0.05）. The results of metabolomics screening proved that the AM mice had 22 intestinal differential metabolites related to oxidative stress，immunity and uterine smooth muscle contraction，such as prostaglandin D₃，glutamic acid，tryptophan，hydroxyeicosatetraenoic acid and stercobilin. Correlation analysis displayed parts of the gut microbiota were significantly correlated with the metabolites. Subsequent experimental verified the levels of reactive oxygen species（ROS），tumor necrosis factor-α（TNF-α）and interleukin-17（IL-17）were significantly higher in AM mice. The GFW treatment demonstrated a significant reduction in expression levels of ROSand TNF-α.

GFW can improve uterine smooth muscle contraction，oxidative stress and immune response in AM mice，which may be related to the regulation of gut microbiota and intestinal metabolites. Its specific regulatory mechanism needs to be confirmed by further studies.