A high-methionine (HM) diet leads to hyperhomocysteinemia (HHcy), while gastrointestinal tissue is an important site of net homocysteine (Hcy) production. However, the role of the gut microbiota in host HHcy remains obscure. This study aimed to determine whether gut microbiota ablation could alleviate host HHcy and glucose intolerance and reveal the underlying mechanism. The results showed that the HM diet-induced HHcy and glucose intolerance in mice, while antibiotic administration decreased the plasma level of Hcy and reversed glucose intolerance. HM diet increased intestinal epithelial homocysteine levels, while antibiotic treatment decreased intestinal epithelial homocysteine levels under the HM diet. Gut microbiota depletion had no effect on the gene expression and enzyme activity of CBS and BHMT in the livers of HM diet-fed mice. The HM diet altered the composition of the gut microbiota with marked increases in the abundances of Faecalibaculum and Dubosiella, which were also positively correlated with plasma Hcy concentrations. An in-depth analysis of the bacterial cysteine and methionine metabolism pathways showed that the abundances of two homocysteine biosynthesis-related KEGG orthologies (KOs) were markedly increased in the gut microbiota in HM diet-fed mice. Hcy was detected from Dubosiella newyorkensis-cultured supernatant by liquid chromatography-tandem mass spectrometry (LC‒MS) analysis. In conclusion, these findings suggested that the HM diet-induced HHcy and glucose intolerance in mice, by reshaping the composition of the gut microbiota, which might produce and secrete Hcy.