Most Aloe species are used as new food or functional food ingredient. Even though widely known for its health benefits, the anti-inflammatory effects and underlying mechanisms of Aloin (Alo), an anthraquinone compound isolated from plant species of the genus Aloe, remain unidentified. Here, we investigated the protective effects of Alo against cecal ligation and puncture (CLP)-induced sepsis and microflora in mice. Alo significantly improved CLP-induced sepsis and the survival rate of septic mice, downregulated the expression of proinflammatory factors, and decreased the infiltration of inflammatory cells in tissues. Alo upregulated the proportion of peritoneal macrophages, reduced the number of peritoneal bacteria, decreased the content of short-chain fatty acids and bile acids in the abdominal cavity, and suppressed Toll-like receptor (TLR)-2/4/nuclear factor kappa-B (NF-κB)/NOD-like receptor thermal protein domain associated protein 3 (NLRP3)/Caspase-1/3/8 signaling. Furthermore, Alo altered the composition of the microbiome and promoted the growth of Lactobacillus, which showed a stronger anti-inflammatory effect. Whole-genome analysis identified the genes Saa3, Il10, Fpr1, and Eif4a1 associated with the protective effects of Alo in mice with CLP-induced sepsis. Overall, our results provide novel insights into the therapeutic potential and mechanism of action of Alo in the treatment of sepsis.