PiaA and PiuA are the lipoprotein components of the Pia and Piu Streptococcus pneumoniae iron uptake ABC transporters and are required for full virulence in mouse models of infection. Active or passive vaccination with recombinant PiuA and PiaA protects mice against invasive S. pneumoniae disease. In this study we have analyzed the antibody responses and mechanism of protection induced by PiuA and PiaA in more detail. For both proteins, two booster vaccinations induced stronger antibody responses in mice than a single or no booster vaccinations, and 5 μg of protein induced similar levels of antibody responses as 20 μg. Immunoglobulin G (IgG) subclass-specific enzyme-linked immunosorbent assays demonstrated that the antibody response to PiuA and PiaA was predominantly IgG1, with induction of only low levels of IgG2a. Anti-PiaA and anti-PiuA polyclonal rabbit antibodies bound to the surface of live S. pneumoniae when assessed by low cytometry but did not inhibit growth of S. pneumoniae in cation-depleted medium or bacterial susceptibility to the iron-dependent antibiotic streptonigrin. However, anti-PiaA and anti-PiuA did increase complement-independent and -dependent opsonophagocytosis of different serotypes of S. pneumoniae by the human neutrophil cell line HL60. Hence, vaccination with PiaA and PiuA protects against S. pneumoniae infection by inducing antibodies that promote bacterial opsonophagocytosis rather than inhibiting iron transport.