Dental caries is a biofilm-mediated disease with Streptococcus mutans as the main pathogenic microorganism, and its incidence is closely related to sucrose. Rubusoside is a natural non-nutritive sweetener isolated from Rubus suavissimu S.Lee. This study was designed to determine the effect of the sucrose substitute on cariogenic properties and virulence gene expression of S. mutans biofilms. S. mutans was exposed to: BHI medium (as a control), BHI+1% sucrose, BHI+1% rubusoside, and BHI +1% xylitol. The growth curve of biofilm was monitored with crystal violet staining and pH was measured every 24 h. After 5 days, the biofilms formed on the glass coverslips were recovered to determine the biomass (dry weight and total soluble proteins), colony-forming units, and intra- and extracellular polysaccharides. Biofilm structural imaging was observed using a scanning electron microscope (SEM). The virulence gene expression (gtfB, gtfC, gtfD, ftf, spaP, gbpB, ldh, atpF, vicR, and comD) was determined by rt-qPCR. Compared to growth in sucrose, xylitol and control, growth in rubusoside resulted in a lower acid production, while also reducing the biofilm accumulation, bacterial viability, and even reducing the production of extracellular polysaccharides. And less biofilm formation and extracellular matrix was observed through SEM in rubusoside than sucrose and xylitol. From the perspective of virulence genes, rubusoside and xylitol significantly inhibited expression of virulence genes compared with sucrose, among which, down-regulation of the gtfB, gtfC, gbpB, ldh, and comD was found in rubusoside than xylitol. Therefore, rubusoside appears to be less potential cariogenicity than sucrose and xylitol, and may become an effective sucrose substitute for caries prevention. Further studies need to deepen these findings. Importance Dental caries, as a major public health challenge, places a heavy biological, social and financial burden on individuals and health care systems. To palliate the deleterious effect of sucrose on the virulence factors of S. mutans, massive commercial products have oriented towards developing sucrose substitutes. Rubusoside, a natural sucrose substitute, is a plant extract with high sweetness. Although some studies have shown that rubusoside do not produce acids and inhibit the growth of S. mutans, little attention has been paid to dental biofilm and underlying mechanisms. Our study focuses on the effect of rubusoside on formation and structure of biofilm, and expression of virulence genes. Results confirms that rubusoside can inhibit accumulation, bacterial viability, polysaccharide production of biofilm, and inhibit related gene expression. These results provide further insight into the cariogenicity of S. mutans biofilm, and demonstrate a new perspective for studying the impact of sucrose substitutes on caries.