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OBJECTIVES The study aimed to evaluate the role of Streptococcus mutans (S. mutans) dexA gene on biofilm structure and microecological distribution in multi-species biofilms. MATERIALS AND METHODS A multi-species biofilm model consisting of S. mutans and its dexA mutants, Streptococcus gordonii (S. gordonii) and Streptococcus sanguinis (S. sanguinis) was constructed, and bacterial growth, biofilm architecture and microbiota composition were determined to study the effect of the S. mutans dexA on multi-species biofilms. RESULTS Our results showed that either deletion or overexpression of S. mutans dexA had no effect on the planktonic growth of bacterium, while S. mutans dominated in the multi-species biofilms to form cariogenic biofilms. Furthermore, we revealed that the SmudexA+ group showed structural abnormality in the form of more fractures and blank areas. The morphology of the SmudexA group was sparser and more porous, with reduced and less agglomerated exopolysaccharides scaffold. Interestingly, the microbiota composition analysis provided new insights that the inhibition of S. gordonii and S. sanguinis was alleviated in the SmudexA group compared to the significantly suppressed condition in the other groups. CONCLUSION In conclusion, deletion of S. mutans dexA gene re-modules biofilm structure and microbiota composition, thereby leading to decreased cariogenicity. Thus, the S. mutans dexA may be an important target for regulating the cariogenicity of dental plaque biofilms, expecting to be a probiotic for caries control. This article is protected by copyright. All rights reserved.