Src family kinases are involved in a plethora of aspects of cellular signaling. We demonstrate in this study that the Src family kinase Lyn negatively regulates TLR signaling in murine bone marrow-derived macrophages (BMM Phis) and in vivo. LPS-stimulated Lyn(-/-) BMM Phis produced significantly more IL-6, TNF-alpha, and IFN-alpha/beta compared with wild type (WT) BMM Phis, suggesting that Lyn is able to control both MyD88- and TRIF-dependent signaling pathways downstream of TLR4. CD14 was not involved in this type of regulation. Moreover, Lyn attenuated proinflammatory cytokine production in BMM Phis in response to the TLR2 ligand FSL-1, but not to ligands for TLR3 (dsRNA) or TLR9 (CpG 1668). In agreement with these in vitro experiments, Lyn-deficient mice produced higher amounts of proinflammatory cytokines than did WT mice after i. v. injection of LPS or FSL-1. Although Lyn clearly acted as a negative regulator downstream of TLR4 and TLR2, it did not, different from what was proposed previously, prevent the induction of LPS tolerance. Stimulation with a low dose of LPS resulted in reduced production of proinflammatory cytokines after subsequent stimulation with a high dose of LPS in both WT and Lyn(-/-) BMM Phis, as well as in vivo. Mechanistically, Lyn interacted with PI3K; in correlation, PI3K inhibition resulted in increased LPS-triggered cytokine production. In this line, SHIP1(-/-) BMM Phis, exerting enhanced PI3K-pathway activation, produced fewer cytokines than did WT BMM Phis. The data suggest that the Lyn-mediated negative regulation of TLR signaling proceeds, at least in part, via PI3K.