Furosine, which is produced when milk and milk products are heated, is bad for people’s health. Furthermore, its content in milk and milk products will increase sharply as the temperature rises. The intestines harbor a highly diverse microbial community, which is critical to maintain health. To investigate furosine’s effect on structure and abundance of gut microflora in ICR mice, a 35-d chronic toxicity model was constructed. Fresh feces sample was collected and detected by Illumina-MiSeq 16s rRNA sequencing technology. We found that the amount of Actinobacteria and Firmicutes in furosine treatment groups increased at the phylum level (P < 0.05); the abundance of Alloprevotella and Bacteroides in furosine treatment groups increased at the genus level (P < 0.05), while the level of Lactobacillus in furosine treatment groups decreased significantly (P < 0.05). In conclusion, furosine increased the complexity and uncertainty of microflora in mice intestine, embodying the downregulation of probiotics and upregulation of maleficent bacteria, indicating that microflora changes might be related to immune injury and intestinal damage caused by long-term intervention of furosine.