Streptococcus parasanguinis is a primary colonizer of dental plaque and an opportunistic pathogen for subacute endocarditis. A putative fibronectin binding protein (Spaf_1409) that lacks both an N-terminal signal peptide and a C-terminal cell wall-anchoring motif was identified from the S. parasanguinis FW213 genome. Spaf_1409 was abundantly present in the cytoplasm and also was found in the cell wall preparation and culture supernatant. By using an isogenic mutant strain, MPH4, Spaf_1409 was found to mediate the binding of S. parasanguinis FW213 to fibronectin. Inactivation of Spaf_1409 did not significantly alter the mass of static biofilm, but reduced the resistance of S. parasanguinis against the shearing force in a flow cell biofilm system, resulting in scattered biofilm. The mortality in Galleria mellonellalarvae infected with MPH4 was higher than in those infected with wild-type S. parasanguinis. However, fewer viable bacterial cells were recovered from larvae infected with MPH4, compared to those infected with wild-typeS. parasanguinis, up to 42 h post infection, suggesting that the infection by MPH4, but not the growth, was responsible for the elevated mortality. The phagocytic analysis using flow cytometry indicated that Spaf_1409 participates in the recognition of S. parasanguinis FW213 by RAW264.7 macrophages, suggesting that inactivation of Spaf_1409 intensified the immune responses in larvae, leading to larval death. Taken together, the data indicate that Spaf_1409 plays different roles in the development of dental biofilm and in systemic infections.