AbstractStreptococcus pneumoniae is responsible for diseases causing major global public health problems, including meningitis, pneumonia and septicaemia. Despite recent advances in antimicrobial therapy, pneumococcal meningitis remains a life-threatening disease. Furthermore, long-term sequelae are a major concern for survivors. Hence, a better understanding of the processes occurring in the central nervous system is crucial to the development of more effective management strategies. We used mass spectrometry based quantitative proteomics to identify protein changes in cerebrospinal fluid from children with Streptococcus pneumoniae infection, compared with children admitted to hospital with bacterial meningitis symptoms but negative diagnosis. Samples were analysed, by label free proteomics, in two independent cohorts (cohort 1: cases (n = 8) and hospital controls (n = 4); cohort 2: cases (n = 8), hospital controls (n = 8)). Over 200 human proteins were differentially expressed in each cohort, of which 65% were common to both. Proteins involved in the immune response and exosome signalling were significantly enriched in the infected samples. For a subset of proteins derived from the proteome analysis, we corroborated the proteomics data in a third cohort (hospital controls (n = 15), healthy controls (n = 5), cases (n = 20)) by automated quantitative western blotting, with excellent agreement with our proteomics findings. Proteomics data are available via ProteomeXchange with identifier PXD004219.