Aims To determine the mechanism of action of antimicrobial protein, lactosporin, against Gardnerella vaginalis and to evaluate its safety in vitro. Methods and Results Bacillus coagulans ATCC 7050 was grown at 37 degrees C for 18 h. The cell-free supernatant was concentrated 10-fold and screened for antimicrobial activity against indicator strain Micrococcus luteus. The mode of action of lactosporin was determined by measuring the potassium release and monitoring the changes in transmembrane potential (Delta psi) and transmembrane pH (Delta pH) of the sensitive cells. Lactosporin caused the efflux of potassium ions from M. luteus cells and dissipation of ?pH in G. vaginalis, while it had no effect on the Delta psi. The safety of lactosporin was evaluated by using EpiVaginal (TM) ectocervical (VEC-100) tissue model. Over 80% of the cells in the vaginal tissue remained viable after exposure to lactosporin for 24 h. Conclusions Lactosporin potentially exerts its antimicrobial activity by selective dissipation of Delta pH and/or by causing leakage of ions from the sensitive cells. Safety studies suggest that lactosporin is a noncytotoxic antimicrobial for vaginal application. Significance and Impact of the Study This study revealed that lactosporin is an effective and safe antimicrobial preparation with potential application for the control of bacterial vaginosis.