Abstract: Background　Female non-obese diabetic (NOD) mice are commonly used for constructing animal models of spontaneous type 1 diabetes mellitus (T1DM), while research on using streptozotocin (STZ) to induce T1DM in male NOD mice is limited, and the impact of STZ on the gut microbiota profile of male NOD mice remains unclear.Objective　To analyze the changes in gut microbiota profiles in STZ-induced T1DM in male NOD mice.Methods　Fourteen male NOD mice were randomly divided into model group (T1DM group, n=8) and control group (CON group, n=6). The mice in the T1DM group were injected with 50 mg/ kg of STZ every day for 5 consecutive days. The mice in the CON group were injected with the same dose of normal saline intraperitoneally every day for 5 consecutive days. The weights and fasting blood glucose of mice were recorded at 0 day, 7 day, and 35 day of modeling. At 35 days, fresh feces were collected for 16S rRNA gene V3-V4 region sequencing to analyze the diversity, composition, differential bacterial screening, and functional prediction of the gut microbiota in male NOD mice with T1DM. Results　Compared with the CON group, the body weight of mice in the T1DM group was significantly reduced at 35 day, and the blood glucose was significantly increased (all P<0.05). The Observed OTUs index, Chao1 index, Simpson index and Shannon index of the gut microbiota of T1DM group were all higher than CON group (all P<0.05). The NMDS analysis (P=0.006, Stress =0.09), PCoA analysis (P=0.003) and PCA analysis (P=0.002) of the gut microbiota in mice of the T1DM group and the CON group showed obvious discrete distances. Compared with the CON group, the Bacteroidota, Patescibacteria, Desulfobacterota, and Cyanobacteria in the gut microbiota of mice in the T1DM group were significantly up-regulated, while the Firmicutes was significantly down regulated (all P<0.05). At the genus level, the Muribaculaceae_unclassified, Muribaculum, Candidatus_Saccharimonas, Desulfovibrio and Alistipes in the T1DM group were significantly up-regulated, while the Ligilactobacillus was significantly down regulated (all P<0.05). The ratios of Firmicutes/Bacteroidota and Firmicutes/Proteobacteria in the gut microbiota of mice in the T1DM group were significantly decreased (all P<0.05). The main differential bacteria Ligilactobacillus and Monoglobus were negatively correlated with fasting blood glucose, while Muribaculum, Alistipes, Candidatus_Saccharimonas, and Eubacterium_siraeum_group were positively correlated with fasting blood glucose (P<0.05). The gut microbiota of T1DM mice undergoes significant changes in nucleotide metabolism, glycolysis metabolism, amino acid metabolism, and lipid metabolism pathways (P<0.05).Conclusion　Streptozotocin induction increases the diversity of gut microbiota and alters its composition in male NOD mice with T1DM. The progression of T1DM in male NOD mice may be associated with streptozotocin-induced structural and functional disturbances in the gut microbiota.