Carrageenan is a common food additive, but its effect on changing the structure of gut microbiota was questioned. In this study, the C57BL/6 mice were given three dosages of λ-carrageenan, including low, medium and high dose (1.7,8.3,41.7mg/kg) for 12 weeks. The effects of λ-carrageenan on glucose metabolism and gut microbiota in mice were investigated by glucose tolerance test, insulin tolerance test and gut microbiota 16S rDNA sequencing. And the role of gut microbiota in the effect of λ-carrageenan on glucose metabolism in mice was verified by fecal bacteria transplantation experiments. The results showed that intake of different dose λ-carrageenan could cause the symptoms of metabolic disorders in mice, such as decreased body weight gain, elevated fasting blood glucose and insulin resistance. By the analysis of gut microbiota composition, it was found that λ-carrageenan could significantly affect the abundance and structure of gut microbiota. At the phylum level, λ-carrageenan increased the relative abundance of the Firmicutes and reduced the relative abundance of Bacteroidota. At the genus level, the abundance of Bacteroidales_S24-7_group_norank was reduced, which regulated blood glucose. The abundance of Lactobacillus and Lachnospira related to lipid metabolism and weight loss was increased by λ-carrageenan administration. And it significantly increased abundance of Akkermansia, which is related to the degradation of the intestinal mucus layer. In addition, the results of fecal bacteria transplantation showed that the metabolic disorders appeared in fecal transplant mice were consistent with those of λ-carrageenan gavage mice. The results showed that intake of λ-carrageenan, especially high doses of λ-carrageenan, induced metabolic disorders in mice by interfering with the composition of the gut microbiota.