A promising alternative vaccine candidate to reduce the burden of pneumococcal diseases is the protein antigen PspA (Pneumococcal surface protein A). Since concomitant colonization with two or more pneumococcal strains is very common in children, we aimed to determine if immunization with PspA would be able to control co-colonization. We evaluated nasal immunization with recombinant PspA (rPspA) in a model of co-colonization with two strains expressing different PspAs. Mice were immunized intranasally with rPspAs from clades 1 to 4 (rPspA1, rPspA2, rPspA3 or rPspA4) using whole-cell pertussis vaccine (wP) as adjuvant. Mice were then challenged with a mixture of two serotype 6B isolates St491/00 (PspA1) and St472/96 (PspA4). Immunization with rPspA1+wP and rPspA4+wP reduced colonization with both strains and the mixture of rPspA1+rPspA4+wP induced greater reduction than a single antigen. Immunization rPspA1+rPspA4+wP also reduced colonization when challenge experiments were performed with a mixture of isolates of serotypes 6B (PspA3) and 23F (PspA2). Furthermore, none of the tested formulations led to a pronounced increase in colonization of one isolate over the other, showing that the vaccine strategy would not favor replacement. Interestingly, the adjuvant wP by itself already led to some reduction in pneumococcal colonization, indicating the induction of non-specific immune responses. Anti-rPspA IgG was observed in serum, nasal wash (NW) and bronchoalveolar lavage fluid (BALF) samples, whereas animals inoculated with formulations containing the adjuvant wP (with or without rPspA) showed higher levels of IL-6 and KC in NW and increase in tissue macrophages, B cells and CD4+T cells in BALF.