Abstract: Objective：To investigate the effect of D-leucine on biofilm formation by Streptococcus mutans.Methods：The Spectrophotometer was used to evaluate the growth curve of Streptococcus mutans exposed to 20.0 mmol/LD-leucine. Construct Streptococcus mutans biofilm model in vitro. Biofilms were formed with 2.5, 5.0, 10.0 and 20.0 mmol/LD-leucine, MTT assay method was used to compare the biofilm formation quantificationally. Confocal laser scanning microscope (CLSM) was applied to observe the biofilm structure after treated with different concentrations of D-leucine. Results：D-leucinedid not affect the growth curves of planktonic S. mutanswithin 24 h. The growth curves attained exponential phase after 3 h andreached stationary phase at 12 h. The quantity of biofilm formed by Streptococcus mutanssignificantlydecreased (P<0.05) and the structure of the biofilm showed by CLSM became thinner with the increase of D-leucine concentration.Conclusions：D-leucinecan inhibit the biofilm formation of Streptococcus mutans.