Toll-like receptor 3 (TLR3) is a vital component of the innate immune system known for its role in recognizing double-stranded RNA (dsRNA), which is commonly associated with viral infections. Within the periodontium, TLR3 is expressed by several cell types, including periodontal ligament cells, gingival fibroblasts, and resident mesenchymal stem/progenitor cells. When activated, TLR3 triggers a series of immunomodulatory responses, such as the upregulation of interferon-gamma (IFN-γ), indoleamine 23-dioxygenase (IDO), and human leukocyte antigen G (HLA-G). These responses collectively influence the local immune environment, promote regulatory T cell differentiation, and modulate the inflammatory process. Crucially, TLR3 activation also plays a significant role in shaping the microenvironment within the periodontium, affecting the regenerative potential of resident mesenchymal stem/progenitor cells. By altering the interactions between these cells and their surrounding environment, TLR3 can either facilitate or hinder tissue repair and regeneration, depending on the disease context. This chapter delves into the complex role of TLR3 in the periodontium, with a particular emphasis on how it influences microenvironmental dynamics in periodontal disease. Understanding these processes opens new avenues for developing targeted therapies that control inflammation, optimize the tissue environment, and enhance periodontal regeneration, ultimately improving the treatment of periodontal disease.