Colonic mucus and gut microbiota closely linked through mutual regulatory effect. While their alterations during sepsis and whether glutamine can maintain the colonic mucus barrier and gut microbiota stability are unclear. Cecal ligation and puncture-induced sepsis in mice was utilized to observe changes in colonic mucus, gut microbiota, and their interaction with glutamine intervention. Our findings indicated that glutamine mitigated sepsis-induced intestinal damage and restores colonic mucus barrier function by augmenting mucin synthesis. Further analysis revealed that goblet cells were under oxidative stress after sepsis, resulting in anterior gradient-2 (AGR2), the key mucin-modifying enzyme, being dissynthesized, and inhibiting mucin 2 (MUC2) maturation. Glutamine could ameliorates this situation by promoting the key enzyme glucose-6-phosphate dehydrogenase (G6PD) glycosylation in the pentose phosphate pathway, increasing the reduced nicotinamide adenine dinucleotide phosphate (NADPH) synthesis, reducing endoplasmic reticulum stress, and accelerating AGR2 synthesis and MUC2 maturation. Additionally, glutamine aided in maintaining gut microbiota stability during sepsis, up-regulating mucin-associated bacteria such as Akkermansia and Alistipes. These bacteria, intimately linked to mucin synthesis and degradation, may impact intestinal mucus stability. In conclusion, glutamine can maintain goblet cell redox balance, promotes AGR2 synthesis and MUC2 maturation, shields the mucus barrier, and potentially maintains gut microbiota stability by regulating the interaction between bacteria and mucus, thus alleviating sepsis-induced intestinal damage.