Considerable evidence suggests that periodontal disease not only is caused by bacterial infection but also is associated with host susceptibility. Using in-house cDNA microarray analysis, we attempted to identify gene expression changes in human periodontal ligament (PDL)-derived cells with and without treatment with lipopolysaccharide (LPS) extracted from Porphylomonas gingivalis (P. gingivalis LPS). Of the five up-regulated genes in the PDLs treated with P. gingivalis LPS, galectin-9, which was reported to have eosinophil chemoattraction, was selected for further analyses. By semiquantitative reverse transcriptase-polymerase chain reaction (sqRT-PCR), real-time quantitative RT-PCR, and Western blot analyses, elevated galectin-9 gene expression was detected in LPS-treated PDL-derived cells. Consequently, it was confirmed that the LPS enhances the expression level of galectin-9 mRNA and protein in a time-dependent manner together with interleukin-8. In addition, strong immunoreaction for galectin-9 was detected in the PDL consisting of the periodontal pocket of a patient with severe periodontal disease. Furthermore, significant up-regulation of galectin-9 mRNA expression was detected in the mRNA from PDLs of patients with periodontal disease when compared with healthy donors (P < 0.05). These results suggest that galectin-9 expression is associated with inflammatory reactions in the PDL.