Type 2 diabetes mellitus (T2DM) is one of the most highly prevalent metabolic disorders worldwide. Uncontrolled T2DM can lead to other health threats such as cardiac arrest, lower-limb amputation, blindness, stroke, impaired kidney function, and microvascular and macrovascular complications. Many studies have demonstrated the association between gut microbiota and diabetes development and probiotic supplementation in improving glycemic properties in T2DM. The study aimed to evaluate the influence ofBifidobacterium brevesupplementation on glycemic control, lipid profile, and microbiome of T2DM subjects. Forty participants were randomly divided into two groups, and they received probiotics (50 × 109CFU/day) or placebo interventions (corn starch; 10 mg/day) for 12 weeks. The changes in the blood-urea nitrogen (BUN), aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), fasting blood sugar (FBS), glycated hemoglobin (HbA1c), total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), creatinine levels, and other factors such as body-mass index, visceral fat, body fat, and body weight were assessed at baseline and after 12 weeks.B. brevesupplementation significantly reduced BUN, creatinine, LDL, TG, and HbA1c levels compared to the placebo group. Significant changes were observed in the microbiome of the probiotic-treated group compared to the placebo group. Firmicutes and proteobacteria were predominant in the placebo and probiotic-treated groups. GeneraStreptococcus,Butyricicoccus, and speciesEubacterium halliiwere significantly reduced in the probiotic-treated group compared to the placebo. Overall results suggested thatB. brevesupplementation could prevent worsening of representative clinical parameters in T2DM subjects. The current study has limitations, including fewer subjects, a single probiotic strain, and fewer metagenomic samples for microbiome analysis. Therefore, the results of the current study require further validation using more experimental subjects.