Streptococcus pneumoniae serotype 3 (Spn3) is still a major cause of disease despite the implementation of the 13-valent pneumococcal conjugate vaccine. We previously reported on the development of an experimental human colonisation Spn3 model and showed that Spn3 was associated with frequent pharyngeal symptoms. Utilising this model, we compared the microbiological colonisation dynamics of Spn3 in the oropharynx and nasopharynx, along with associated symptoms and polysaccharide antibody responses. A total of 96 healthy adults (aged 18–50 years) were nasally inoculated with different dosages and strains of Spn3. Pneumococcal carriage frequency and density post-challenge were determined using culture and multiplex qPCR from nasal wash and saliva samples collected on days 2, 7, and 14 post-challenge. Spn3 density was compared with historical Spn6B data at day 2 post-challenge. IgG antibodies recognising the Spn3 capsular polysaccharide were measured at baseline and day 14 post-challenge in both niches. Spn3 colonisation frequency was found to be similar in the nasopharynx and oropharynx on any day post-challenge, independently of challenge dose or strain tested. In saliva, Spn3 density was greatest at a challenge dose of 80,000 CFU/naris compared to the lowest or highest exposure doses. Of note, on day 2 post-challenge, Spn3 density was significantly higher than that observed following the Spn6B challenge at the same dose (p = 0.00090). No significant difference in Spn3 density was observed for challenge strain or symptomology on any day post-challenge. Spn3 polysaccharide-specific salivary IgG levels increased from baseline to day 14 in colonised participants (p = 0.0051) with a median IgG fold change of 1.458 [IQR 0.6131–4.281], but no increase in IgG levels occurred in uncolonized participants (p = 0.19). Participants colonised with the Spn3 clade Iα but not clade II had significant increases in polysaccharide-specific IgG from baseline (p = 0.0081). Given equivalent exposure doses, Spn3 colonised the oropharynx at higher densities than Spn6B. These data could help explain continued Spn3 population transmission despite widespread use of a Spn3-containing pneumococcal conjugate vaccine in the UK paediatric immunization programme. Within Spn3, clade Iα but not clade II increased mucosal IgG response following colonisation, which could help explain increasing transmission of clade II in the UK and higher transmission of this clade in mouse models.