Toll-like receptors (TLRs) sense invading microbial pathogens and play crucial roles in the activation of innate and adaptive immunity. However, excessive TLR activation can disrupt immune homeostasis, and may be responsible for the development of autoimmune and inflammatory diseases. As such, the molecules and pathways that negatively control TLR signaling have been intensively investigated. Here, we discuss recent insights into the negative regulation of TLR signaling, with focus on three major mechanisms: (i) dissociation of adaptor complexes; (ii) degradation of signal proteins; and WO transcriptional regulation. We also highlight how pathogens negatively target TLR signaling as a strategy to evade the host immune response.