Aim: The alpha-glucosidase inhibitor acarbose is approved for the treatment of type 2 diabetes (T2D). It acts in the lumen of the gut by reducing intestinal hydrolysis and absorption of ingested carbohydrates. This reduces postprandial blood glucose concentration and increases the content of carbohydrates in the distal parts of the intestine potentially influencing gut microbiome (GM) composition and possibly impacting the gut microbiome (GMdysbiosis associated with T2D. Here, we investigated the effect of acarbose on GM composition in patients with T2D Methods: Faecal samples were collected in a previously conducted randomised, placebo-controlled, double-blind, crossover study in which 15 individuals with metformin-treated T2D (age 57–85 years, HbA1c 40–74 mmol/mol, BMI 23.6–34.6 kg/m²) were subjected to two 14-day treatment periods with acarbose and placebo, respectively, separated by a 6-week wash-out period. Faecal samples were collected before and by the end of each treatment period. The GM profiles were evaluated by 16S rRNA gene amplicon sequencing. Results: The GM profiles after the treatment periods with acarbose or placebo remained unaffected (P > 0.7) when compared with the GM profiles before treatment. This applied to the analysis of within-sample diversity (α-diversity) and between-sample bacterial composition diversity (β-diversity). Additionally, no dominant bacterial species differentiated the treatment groups, and only minor increases in the relative abundances of Klebsiella spp. and Escherichia coli (P < 0.05) were observed after acarbose treatment. Conclusion: In patients with metformin-treated T2D, 14 days of treatment with acarbose showed only minor effects oGM as seen in increased relative abundances of Klebsiella spp. and Escherichia coli.