Streptococcus pneumoniae is a pathogenic human bacterium responsible for more deaths worldwide than any other single pathogen, yet it is more commonly carried asymptomatically as part of the human commensal nasopharyngeal flora. The mechanisms underlying pneumococcal transition from commensal to pathogen are poorly understood, largely due to its vast genetic diversity. S. pneumoniae is subdivided into >90 serotypes, superimposed on >5000 clonal types distinguished by sequence type (ST). Previous studies showed strains within the same clonal lineage display distinct niche adaptation in mice, depending on their site of isolation. Serotype 14 (ST15) clinical isolates derived from the blood (including strain 4559) infected the lungs, while ear isolates (including strain 947) infected the ear and brain. Genomic comparisons performed between 4559 and 947 identified a single nucleotide polymorphism (SNP) in rafR, which encodes a protein involved in the uptake of the trisaccharide raffinose. Growth assays with raffinose as the sole carbon source in chemically defined media showed that 4559 grew at a greater rate and to a higher density than 947, whereas there was no difference in growth in the presence of glucose. Finally, the rafR alleles were swapped by allelic-exchange mutagenesis between 4559 and 947 to generate 4559947rafR and 9474559rafR. These mutants as well their respective wild types were then used to infect 8 mice intranasally for each strain. At 24h post intransal challenge, 4559947rafR was present in significantly less numbers than 4559 in the lungs, yet in significantly higher numbers in the ear and brain. 9474559rafR was present in significantly higher numbers than 947 in the lungs, and in significantly lower numbers in the ear and brain. This virulence phenotype switch suggests that the raffinose pathway plays a significant role in dictating pneumococcal disease progression, which could be a potential target for novel vaccines or treatments.