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Toll-like receptors (TLRs) are critical pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs), which are conserved and specific molecular “signatures” expressed by pathogens. TLR ligation triggers distinct but shared signaling pathways that lead to effector mechanisms in innate immune responses. TLR specificity and activation are strictly and finely tuned at multiple levels of various signal transduction pathways, resulting in complex signaling platforms. Many molecules, ranging from membrane and cytosol to nuclear, contribute to TLR ligand discrimination or receptor signaling and play different roles in the regulation of TLR responses via different mechanisms, such as cross-regulation, protein modification, helper cofactors, and posttranscriptional and epigenetic regulation. Herein, we summarize the most recent literature that provides new insight into regulation of TLR signaling-triggered innate immune responses. A greater understanding of the mechanisms underlying the control of TLR signaling may provide new targets for therapeutic intervention for infections and inflammatory diseases.