Streptococcus sanguinis is an early colonizer of the tooth surface and competes with oral pathogens such as Streptococcus mutans to maintain oral health. However, little is known about its mechanism of biofilm formation. Here, we show that mutation of the ciaR gene, encoding the response regulator of the CiaRH two-component system in S. sanguinis SK36, produced a fragile biofilm. Cell aggregation, gtfP gene expression and water-insoluble glucan production were all reduced, which suggested polysaccharide production was decreased in ΔciaR. RNA sequencing and qRT-PCR revealed that arginine biosynthesis genes (argR, argB, argC, argG, argH and argJ) and two arginine/histidine permease genes (SSA_1568 and SSA_1569) were upregulated in ΔciaR. In contrast to ΔciaR, most of strains constructed to contain deletions in each of these genes produced more biofilm and water-insoluble glucan than SK36. A ΔciaRΔargB double mutant was completely restored for the gtfP gene expression, glucan production and biofilm formation ability that was lost in ΔciaR, indicating that argB was essential for ciaR to regulate biofilm formation. We conclude that by promoting the expression of arginine biosynthetic genes, especially argB gene, the ciaR mutation reduced polysaccharide production, resulting in the formation of a fragile biofilm in Streptococcus sanguinis.