Porphyromonas gulae, a Gram-negative black-pigmented anaerobe, has been associated with periodontal disease in companion animals and its virulence has been attributed to various factors, including lipopolysaccharide (LPS), protease and fimbriae. Toll-like receptors (TLRs) recognise pathogen-associated molecular patterns, such as peptidoglycan, lipids, lipoproteins, nucleic acid and LPS. Following P. gulae infection, some inflammatory responses are dependent on both TLR2 and TLR4. In addition, a recent clinical study revealed that acute and persistent inflammatory responses enhance the expressions of TLR2 and TLR4 in the oral cavity. In this study, we investigated the interaction between P. gulae LPS and human gingivalis epithelial cells (Ca9-22 cells). P. gulae LPS was found to increase TLR2 and TLR4 mRNA expressions and protein productions, and enhanced inflammatory responses, such as COX2 , TNF-ɑ, IL-6 and IL-8. Stimulated Ca9-22 cells exhibited phosphorylation of ERK1/2 and p38, and their inhibitors diminished inflammatory responses, while knockdown of the TLR2 and/or TLR4 genes with small interfering RNA (siRNA) prevented inflammatory responses. Moreover, p38 and ERK1/2 phosphorylation was decreased in TLR2 and TLR4 gene knockdown cells. These findings suggest that P. gulae LPS activates p38 and ERK1/2 via TLR2 and TLR4, leading to inflammatory responses in human gingival epithelial cells.