The lipopolysaccharide (LPS) secreted by Porphyromonas gingivalis is implicated in the initiation and progression of periodontitis. Human gingival fibroblasts (HGFs) are the major constituent of gingival connective tissue. In this study, we examined the expression of Toll-like receptor 4 (TLR4) on HGFs by flow cytometric analysis, and studied the signal transduction induced by LPS stimulation of HGFs by enzyme-linked immunosorbent assay, Western blotting, and immunoprecipitation. We show that LPS binds to HGFs, and that HGFs express TLR4 and myeloid differentiation primary response gene 88 (MyD88). P. gingivalis LPS-induced interleukin (IL)-1 production in HGFs was inhibited by anti-TLR4 antibody. P. gingivalis LPS treatment of HGFs activated several intracellular proteins including protein tyrosine kinases, and upregulated the expression of IL-1 receptor-associated kinase (IRAK), nuclear factor-kappa B (NF-kappa B), and activating protein-1 (AP-1), and these events were suppressed by anti-TLR4 monoclonal antibody. Our findings suggest that the binding of P. gingivalis LPS to TLR4 on HGFs activates various second messenger systems.