AbstractLower respiratory tract infections caused by Streptococcus pneumoniae (Spn) are a leading cause of death globally. Here we investigate the bronchial epithelial cellular response to Spn infection on a transcriptomic, proteomic and metabolic level. We found the NAD⁺ salvage pathway to be dysregulated upon infection in a cell line model, primary human lung tissue and in vivo in rodents, leading to a reduced production of NAD⁺. Knockdown of NAD⁺ salvage enzymes (NAMPT, NMNAT1) increased bacterial replication. NAD⁺ treatment of Spn inhibited its growth while growth of other respiratory pathogens improved. Boosting NAD⁺ production increased NAD⁺ levels in immortalized and primary cells and decreased bacterial replication upon infection. NAD⁺ treatment of Spn dysregulated the bacterial metabolism and reduced intrabacterial ATP. Enhancing the bacterial ATP metabolism abolished the antibacterial effect of NAD⁺. Thus, we identified the NAD⁺ salvage pathway as an antibacterial pathway in Spn infections, predicting an antibacterial mechanism of NAD⁺.