Streptococcus pneumoniae is the most common cause of bacterial illness worldwide, causing more than 1.5 million deaths annually. S. pneumoniae commonly colonises the nasopharynx of healthy individuals asymptomatically. A complete understanding of the mechanism(s) involved in pneumococcal pathogenesis is lacking. Current vaccines against S. pneumoniae (PCV-13 and PPSV-23) remain ineffective against untargeted strains. A complete understanding of the pathobiology of S. pneumoniae will aid development of more effective vaccines and treatments. Our recent work reports a novel, randomly switching, N6-adenosine DNA methyltransferase (the SpnD39III system) producing six different specificities (alleles A-F). These variants produce six distinct phenotypes via epigenetic regulation of multiple genes; a phasevarion. We sought to characterize the phenotypes of the six SpnD39III alleles by examining the effect of SpnD39II switching to clinically relevant traits such as survival in human blood, biofilm formation, adherence and invasion, and capsule production. We evaluated if SpnD39III allele switching occurred in vivo (mice) across different host niches. The effect of SpnD39III phase-variation on current protein vaccine candidate expression was also investigated using RT-qPCR and Western Blot methodologies. Analysis showed that SpnD39III switching influences multiple differences in phenotype in vitro. In vivo analysis suggests selection for/against particular SpnD39III in different host niches. Significant differences in putative vaccine gene/protein target expression was also influenced by SpnD39III phase-variation. This data provides a robust understanding of gene regulation and pathobiology influenced by phasevarion switching, and will inform and direct future vaccine development against this major human pathogen.