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The aim of this study was to analyze the differences in gut microbiota between selenium deficiency and T-2 toxin intervention rats. Knee joint and fecal samples of rats were collected. The pathological characteristics of knee cartilage were observed by safranin O/fast green staining. DNA was extracted from fecal samples for PCR amplification, and 16S rDNA sequencing was performed to compare the gut microbiota of rats. At the phylum level, <i>Firmicutes</i> (81.39% vs. 77.06%) and <i>Bacteroidetes</i> (11.11% vs. 14.85%) were dominant in the Se-deficient (SD) group and T-2 exposure (T-2) groups. At the genus level, the relative abundance of <i>Ruminococcus_1</i> (12.62%) and <i>Ruminococcaceae_UCG-005</i> (10.31%) in the SD group were higher. In the T-2 group, the relative abundance of <i>Lactobacillus</i> (11.71%) and <i>Ruminococcaceae_UCG-005</i> (9.26%) were higher. At the species level, the high-quality bacteria in the SD group was <i>Ruminococcus_1_unclassified</i>, and <i>Ruminococcaceae_UCG-005_unclassified</i> in the T-2 group. <i>Lactobacillus_sp__L_YJ</i> and <i>Lactobacillus_crispatus</i> were the most significant biomarkers in the T-2 group. This study analyzed the different compositions of gut microbiota in rats induced by selenium deficiency and T-2 toxin, and revealed the changes in gut microbiota, so as to provide a certain basis for promoting the study of the pathogenesis of Kashin–Beck disease (KBD).