Toll-like receptor (TLR) 4 recognition of lipopolysaccharide (LPS) triggers signalosome assembly among TLR4, sorting (e.g., MyD88-adapter-like, Mal) and signaling (e.g., MyD88) adapters, initiating recruitment and activation of kinases, activation of transcription factors and production of inflammatory mediators. In this study, we examined whether tyrosine phosphorylation of Mal regulates its interactions with TLR4, MyD88, IRAK-2, TRAF-6 and is important for signaling. Overexpression of wild-type (WT) Mal in human embryonic kidney (HEK) 293T cells induced its constitutive tyrosine phosphorylation and led to activation of p38, NF-B, and IL-8 gene expression. Mutagenesis of Y86, Y106, and Y159 tyrosine residues within the Toll-IL-1R (TIR) domain impaired Mal tyrosine phosphorylation, interactions with Bruton tyrosine kinase (Btk), phosphorylation of p38, and NF-B activation. LPS triggered tyrosine phosphorylation of endogenous Mal and initiated Mal-Btk interactions in 293/TLR4/MD-2 cells and human monocytes that were suppressed in endotoxin-tolerant cells. Compared to WT-Mal, Y86A-, Y106A-, and Y159A-Mal variants exhibited higher interactions with TLR4 and MyD88, while associations with IL-1R-associated kinase (IRAK)-2 and TNFR-associated factor (TRAF)-6 were not affected. Overexpression of Y86A and Y106A Mal in 293/TLR4/MD-2 cells exerted dominant-negative effects on TLR4-inducible p38 phosphorylation and NF-B reporter activation to the extent comparable to P125H Mal-mediated suppression. In contrast, tyrosine-deficient Mal species did not affect NF-B activation when signaling was initiated at the post-receptor level by overexpression of MyD88, IRAK-2 or TRAF-6. Thus, tyrosine phosphorylation of Mal is required for adapter signaling, regulates Mal interactions with TLR4 and receptor signaling, and is inhibited in endotoxin tolerance.