Background: Selective sodium-glucose cotransporter 2 inhibitors, known to lower the blood glucose levels by promoting the urinary glucose excretion, can predispose to genitourinary infections. This prospective study investigated the influence of selective sodium-glucose cotransporter 2 inhibitors luseogliflozin on the vaginal flora of the pre- and postmenopausal women with type 2 diabetes mellitus. Methods: Twelve premenopausal and 24 postmenopausal female Japanese patients with type 2 diabetes mellitus took luseogliflozin 2.5 mg once daily for 6 months. The intravaginal fungal and bacterial populations, together with the body weight and serum parameters of diabetes mellitus and lipid metabolism were measured before and after the treatment. Results: After luseogliflozin treatment, the body weight, body mass index and hemoglobin A1c decreased, and the serum levels of high-density lipoprotein cholesterol increased significantly. Luseogliflozin treatment revealed to increase vaginal colony concentrations of Enterococcus faecalis (P = 0.0077) and E. coli (P = 0.0201) in premenopausal patients, and Enterococcus faecalis (P = 0.0051) and Candida albicans (P = 0.0355) in postmenopausal patients. In both pre- and postmenopausal patients, colony concentrations of Staphylococcus spp . had decreased (P = 0.0261 and P = 0.0161). Conclusions: Treatment with selective sodium-glucose cotransporter 2 inhibitors luseogliflozin was associated with changes of the vaginal flora. These findings provide basic data on the increased susceptibility to genital infections during luseogliflozin treatment. J Clin Med Res. 2021;13(5):309-316 doi: https://doi.org/10.14740/jocmr4504