OBJECTIVES: Colonisation of the upper airways by Streptococcus pneumoniae is a prerequisite for disease development and shapes immune responses that vary between geographical settings. This first controlled human pneumococcal infection study in the Netherlands assessed colonisation, antibody responses, respiratory symptoms, and concurrent viral infections. METHODS: Twenty healthy adults (19-47 years) were enrolled between May 2022 and February 2023 and inoculated intranasally with 160,000 colony forming units of S. pneumoniae serotype 6B (strain BHN418). Participants were monitored for 28 days to assess colonisation, respiratory viral infection and safety. Serum and nasal lining fluid were collected to measure IgA, IgM and IgG responses to serotype 6B polysaccharide (6B) and whole pneumococci. RESULTS: Eight of 20 participants (40%) became colonised. Colonised participants showed significant rises in serum and mucosal IgG and IgA to 6B, but not to pneumococci. Fifteen participants tested positive for at least one respiratory virus. Symptoms were generally mild but occurred more often in those with concurrent pneumococcal colonisation and viral infection. CONCLUSIONS: Colonisation rates were comparable to studies in other populations. Colonisation induced systemic and mucosal antibody responses, while viral co-infection increased symptom burden. This controlled infection study demonstrates feasibility and safety, and establishes a platform for vaccine evaluation and pneumococcal-viral interaction studies.