AbstractStreptococcus pneumoniae is a causative agent of community‐acquired pneumonia. Upon pneumococcal infection, innate immune cells recognize pneumococcal lipoproteins via Toll‐like receptor 2 and induce inflammation. Here, we generated a strain of S. pneumoniae deficient in lipoprotein signal peptidase (LspA), a transmembrane type II signal peptidase required for lipoprotein maturation, to investigate the host immune response against this strain. Triton X‐114 phase separation revealed that lipoprotein expression was lower in the LspA‐deficient strain than in the wild‐type strain. Additionally, the LspA‐deficient strain decreased nuclear factor‐κB activation and cytokine production in THP‐1 cells, indicating impaired innate immune response against the strain.