Small salivary phosphoproteins – statherin (ST) and histatin 1 (HT1) – are found in the acquired enamel pellicle which modulates Streptococcus mutans adhesion onto dental enamel. However, their roles in S. mutans adhesion onto enamel surfaces are still undefined. The aim of this study was to investigate whether and how ST and HT1 affect (i) S. mutans adhesion and (ii) the adsorption of S. mutans adhesion-promoting salivary proteins onto hydroxyapatite (HA) in vitro. We fractionated human parotid saliva by adsorption to HA and further by gel filtration chromatography. Adhesion of [3H]-labeled S. mutans strain MT8148 onto sintered HA plates was promoted significantly (>10-fold) by high-molecular weight glycoprotein fraction (HMWGP), but not by purified ST or HT1. More interestingly, promotion of S. mutans adhesion onto HA by HMWGP was significantly reduced by adding purified ST or HT1 to HMWGP. [3H]-labeled S. mutans adhesion on HA was positively correlated to the [14C]-labeled HMWGP adsorption onto HA, which was also reduced by the addition of purified ST and HT1. Synthetic peptides corresponding to ST and HT1 reduced the parotid saliva-promoted S. mutans adhesion. However, removal of the negative charges in the N-terminal domains of ST and HT1 diminished their inhibitory effects on S. mutans adhesion promoted by parotid saliva. We conclude that ST and HT1 competitively inhibit the adsorption of salivary HMWGP, and thereby reduce S. mutans adhesion onto HA surfaces.