Toll-like receptors (TLRs) are pivotal components of the innate immune response, which is responsible for eradicating invading microorganisms through the induction of inflammatory molecules. These receptors are also involved in responding to harmful endogenous molecules and have crucial roles in the activation of the innate immune system and shaping the adaptive immune response. However, TLR signaling pathways must be tightly regulated because undue TLR stimulation may disrupt the fine balance between pro- and anti-inflammatory responses. Such disruptions may harm the host through the development of autoimmune and inflammatory diseases, such as rheumatoid arthritis and systemic lupus erythematosus. Several studies have investigated the regulatory pathways of TLRs that are essential for modulating proinflammatory responses. These studies reported several pathways and molecules that act individually or in combination to regulate immune responses. In this review, we have summarized recent advancements in the elucidation of the negative regulation of TLR signaling. Moreover, this review covers the modulation of TLR signaling at multiple levels, including adaptor complex destabilization, phosphorylation and ubiquitin-mediated degradation of signal proteins, manipulation of other receptors, and transcriptional regulation. Lastly, synthetic inhibitors have also been briefly discussed to highlight negative regulatory approaches in the treatment of inflammatory diseases.