Abstract Type 2 diabetes (T2D) is a chronic disease that manifests with endocrine and metabolic disorders, seriously threatening public health. This study aims to investigate the effects of Bacillus sp. DU-106 on anti-diabetic and gut microbiota in C57BL/6J mice on a high-fat diet (HFD) and streptozotocin (STZ)-induced T2D. Bacillus sp. DU-106 was administered to model mice for 8 consecutive weeks. Oral administration of Bacillus sp. DU-106 led to decreased food and water intake and alleviated body weight loss. And Bacillus sp. DU-106 imparted several health benefits to the mice, including balanced blood glucose, alleviation of insulin resistance in the T2D mice, and an improvement in lipid metabolism. Furthermore, Bacillus sp. DU-106 protected against liver and pancreas impairment to some extent. Additionally, Bacillus sp. DU-106 treatment reshaped the intestinal flora by enhancing gut microbial diversity and enriching the abundance of certain functional bacteria. Collectively, these findings suggested that Bacillus sp. DU-106 has the potential to ameliorate T2D by regulation of the gut microbiota. Therefore, a novel probiotic proposed here, Bacillus sp. DU-106 might be a promising therapeutic agent to prevent and alleviate T2D in mice.