Interaction between surfactant protein-A (SP-A) and toll-like receptor (TLR)4 plays a critical role in host defense. In this work， we studied the host defense function of SPA4 peptide (amino acids GDFRYSDGTPVNYTNWYRGE)， derived from the TLR4-interacting region of SP-A， against Pseudomonas aeruginosa. We determined the binding of SPA4 peptide to live bacteria， and its direct antibacterial activity against P. aeruginosa. Pro-phagocytic and anti-inflammatory effects were investigated in JAWS II dendritic cells and primary alveolar macrophages. The biological relevance of SPA4 peptide was evaluated in a mouse model of acute lung infection induced by intratracheal challenge with P. aeruginosa. Our results demonstrate that the SPA4 peptide does not interact with or kill P. aeruginosa when cultured outside the host. The SPA4 peptide treatment induces the uptake and localization of bacteria in the phagolysosomes of immune cells. At the same time， the secreted amounts of TNF-α are significantly reduced in cell-free supernatants of SPA4 peptide-treated cells. In cells overexpressing TLR4， the TLR4-induced phagocytic response is maintained， but the levels of TLR4-stimulated TNF-α are reduced. Furthermore， our results demonstrate that the therapeutic administration of SPA4 peptide reduces bacterial burden， inflammatory cytokines and chemokines， intracellular signaling， and lactate levels， and alleviates lung edema and tissue damage in P. aeruginosa-infected mice. Together， our results suggest that the treatment with SPA4 peptide can help control the bacterial burden， inflammation， and tissue injury in a P. aeruginosa lung infection model.