Resident alveolar macrophages and respiratory epithelium constitutes the first line of defense against invading lung pneumococci. Results from our study showed that increased mortality and bacterial outgrowth and dissemination seen in morphine-treated mice were further exaggerated following depletion of alveolar macrophages with liposomal clodronate. Using an in vitro alveolar macrophages and lung epithelial cells infection model, we show significant release of MIP-2 from alveolar macrophages, but not from lung epithelial cells, following 4 h of exposure of cells to pneumococci infection. Morphine treatment reduced MIP-2 release in pneumococci stimulated alveolar macrophages. Furthermore, morphine treatment inhibited Streptococcus pneumoniae-induced NF-κB-dependent gene transcription in alveolar macrophages following 2 h of in vitro infection. S. pneumoniae infection resulted in a significant induction of NF-κB activity only in TLR9 stably transfected HEK 293 cells, but not in TLR2 and TLR4 transfected HEK 293 cells, and morphine treatment inhibited S. pneumoniae-induced NF-κB activity in these cells. Moreover, morphine treatment also decreased bacterial uptake and killing in alveolar macrophages. Taken together, these results suggest that morphine treatment impairs TLR9-NF-κB signaling and diminishes bacterial clearance following S. pneumoniae infection in resident macrophages during the early stages of infection, leading to a compromised innate immune response.