Background This study aimed to confirm the existence of gut microbiota (GM) imbalance in the early stage of sepsis, observe the effect of fecal microbiota transplantation (FMT) on sepsis, and explore whether FMT can reconstruct the GM of septic mice and restore its protective function on the intestinal mucosal barrier. Methods The study included acute experiments and 7-day mortality observation experiments with clean-grade C57BL/6, and they were randomly divided randomly into three groups, namely, the sham group, the sepsis model group and the fecal microbiota transplantation group. Fresh feces from 10 mice were kept every day to make fecal liquid. The Sham group and the CLP group were given intragastric administration once a day with phosphate-buffered saline, and the FMT group mice were given fecal microbiota transplantation once a day. The animals were euthanized at 12, 24, and 48 h after modeling, and blood, colon, and stool from each mouse were collected at the same time.Results Colonic pathological scores, pro-inflammatory cytokines, TLR4/MyD88/NF-κB protein levels, and gene expression levels, were lower in the FMT group, while anti-inflammatory factors, mucus layer thickness, MUC2, occludin, and ZO-1 proteins were higher in the FMT group than the CLP group. Bacterial flora analysis showed gut flora was reconstructed after FMT. The species composition of the differential pathways revealed that the Lachnospiraceae group contributed the most by the L-lysine pathway of fermentation to acetate and butanoate.Conclusion GM imbalance exists early in sepsis. FMT can improve morbidity and effectively reduce mortality in septic mice. After the fecal bacteria were transplanted, the abundance and diversity of the gut flora were restored, and the microbial characteristics of the donors changed. FMT can effectively reduce epithelial cell apoptosis, improve the composition of the mucus layer, upregulate the expression of tight junction proteins, and reduce intestinal permeability and the inflammatory response, thus protecting the intestinal barrier function. After FMT, Lachnospiraceae contributes the most to intestinal protection through enhancement of the L-lysine fermentation pathway, resulting in the production of acetate and butanoate, and may be the key bacteria for short-chain fatty acid metabolism and FMT success.