Background MMP8 inhibitors may serve as potential therapeutic targets for sepsis. Currently, there are limited domestic studies investigating the effects of MMP8 inhibitors on the treatment of sepsis and their impact on the gut microbiome of mice. Objective This study aims to investigate the compositional characteristics of the gut microbiome in septic mice, identify differential microbiome, and evaluate the impact of MMP8 inhibitors on the gut microbiome of these septic mice. Methods Twenty 6-week-old SPF grade C57BL/6J mice were randomly assigned to four groups: model, treatment, sham operation, and control, with five mice in each group. In the treatment and model group, the abdominal cavity was opened to expose the cecum, which was then ligated and punctured at its midpoint to create a sepsis model. Mice in the sham operation group underwent the same abdominal procedure, but the cecum was not ligated or punctured, and the cavity was closed afterward. Twenty-four hours prior to modeling, the treatment and sham operation groups received intraperitoneal injections of 0.3 mg/kg MMP8 inhibitor every 12 hours, while the control and model groups were administered an equal volume of PBS buffer. All mice were sacrificed 24 hours postsurgery, and the colon contents from each group were collected for sequencing of the V3-V4 region of the 16S rRNA gene. Subsequently, the α and β-diversity, as well as the gut microbiome composition of the each group, were analyzed to assess changes in differential microbiome composition and diversity. Results Compared with the model group, the number of ASVs (P=0.029), Observed species index (P=0.029), Chao1 index (P=0.028), and Shannon index (P=0.016) in the treatment group after treatment were significantly increased. PCA(P=0.001), PCoA(P=0.001) and NMDS(P=0.001,Stress=0.13) analysis results showed that the gut microbiome composition of the treatment group and the model group was far away from and had no overlap with the control and sham operation group. The gut microbiome composition of the treatment group partially overlapped with that of the model group mice after treatment. The main bacterial genera in treatment group were Muribaculaceae-unclassified, Escherichia-Shigella, Enterobacter; the main bacterial genera in the model group were Ligilactobacillus, Escherichia-Shigella, and Enterobacter. LEfSe analysis found that the differential bacteria in the treatment group were Enterobacter and Klebsiella, and the model group was Escherichia-Shigella. Compared with the sham operation group, the ratio of Firmicutes to Proteobacteria was reduced in the treatment (P=0.001) and model groups (P=0.001). Compared with the model group, the gut microbiome of the mice in the treatment group were up-regulated in metabolic pathways dominated by Amino acid related enzymes and Cell cycle-Caulobacter, and downregulated in metabolic pathways dominated by Glycosyltransferases and Bacterial secretion system. Conclusion In septic mice, there was a significant increase in harmful bacteria within the intestines, accompanied by a reduction in the diversity of intestinal flora. However, the intervention with an MMP8 inhibitor notably improved the diversity of gut microbiome in these septic mice, leading to an increase in beneficial bacteria and a decrease in harmful bacteria. This finding is promising, suggesting that MMP8 inhibitors could serve as potential therapeutic agents for ameliorating gut microbiome disorders associated with sepsis.