Toll-like receptors (TLRs) are a family of highly conserved pattern recognition receptors that can recognize both pathogen-associated molecular patterns and danger-associated molecular patterns. These receptors are important in the activation of the innate immune system and play a role in shaping the adaptive immune system. For years, the expression of TLRs in the brain has been proposed to contribute to the immunological protection of the central nervous system. However, emerging studies have provided increasing evidence of non-immune functions of TLRs and suggest that these receptors may participate in more complex processes that extend beyond the regulation of the innate immune response. In this review, we highlight the expression of TLRs in non-immune cells and epitomize TLR non-immune functions. We aim to reveal the novel roles of TLRs that are distinct from their traditional functions in immunity. Negative regulatory approaches used to study TLR signaling pathways are also discussed, providing potential directions for further studies.