Macrophages are armed with Toll-like receptors (TLRs) for detecting and eliminating danger signals. Activation of TLRs results in classical activation (M1 polarization) of macrophages and triggers pro-inflammatory responses, then also resolves inflammation after clearing the danger. Class IB phosphatidylinositol 3-kinase (PI3K), PI3Kγ, has emerged as a key determinant of macrophage programming and polarization. Our lab has previously defined how in M1 cells, activated by TLRs, the LRP1-Rab8a-PI3Kγ complex cross-talking with TLRs, acts to modulate Akt/mTOR signaling and cytokine outputs that are biased towards an anti-inflammatory axis to help suppress inflammation. PI3Ks have closely aligned functions with members of the Src family kinases (SFKs). Lyn is one such SFK, which has known and varied roles in modulating macrophage programming and inflammatory status. In this study, through GST pull-down assays, mass spectrometry analysis and co-immunoprecipitation assays, we have revealed a novel interaction between Lyn and PI3Kγ in TLR pathways. Using Lyn KO BMMs and PI3Kγ specific inhibitor, we showed Lyn negatively regulates PI3Kγ/Akt signaling and promotes expression of pro-inflammatory mediators. Furthermore, we have also pinpointed regulatory SH3 domain of Lyn to be responsible for interaction with the catalytic subunit of PI3Kγ. Live cell imaging revealed macropinosomes as the location for Lyn/PI3Kγ interaction. These results reveal new complexities and insights for modulating Akt signaling and macrophage programming through the involvement of Lyn and PI3Kγ.