Lyn is an important B cell signaling kinase of the Src tyrosine kinase family with a broad range of functions from cytoskeletal changes to induction of apoptosis. However, the role of Lyn in infectious diseases is not clear. Here, we demonstrate that Lyn activation by phosphorylation significantly impacted invasion of an alveolar epithelial cell line, primary lung cells, and rat lungs by Pseudomonas aeruginosa (PA), a common opportunistic lung pathogen affecting individuals with deficient lung immunity. Our results indicate that activation of Lyn and its interaction with rafts and TLR2, played an important role in the initial stages of PA interaction with host cells. The role of Lyn was further evaluated using the pharmacologic Src-specific inhibitor PP2, a dominant negative mutant, and finally confirmed with Lyn-deficient (Lyn–/–) bone marrow-derived mast cells. Inhibition of Lyn's function by above approaches prevented PA internalization. Moreover, blocking of Lyn also affected downstream events: induction of inflammatory cytokines and apoptosis. This report brings out a new role of Lyn in infectious diseases and indicates potential new targets for prevention and treatment of infections.