Zingiber cassumunar Roxb. was previously shown to possess anti-tumor promoter and potent anti-inflammatory activities. Objective: The aim of this study was to investigate the effect of its ethanol extract on the production of hyaluronan (HA), which is an inflammatory marker, in cultured human gingival fibroblasts. Methods: Cultured fibroblasts were either treated with 0.1-50 μg/ml of the extract, 0.1-50 μM of retinoic acid (RA), or 1 μg/ml of 12-O-tetradecanoyl phorbol-13-acetate (TPA) for indicated times, or left untreated as a control. Culture medium was analyzed for HA quantities by the ELISA-based assay. Moreover, total RNA was harvested and RT-PCR analyses were performed according to a standard protocol to determine mRNA expression of hyaluronan synthase (HAS) -1, -2, and –3. Results: The extract of Zingiber cassumunar decreased HA levels in culture medium in a dose-dependent manner. In addition, combination between RA and Zingiber cassumunar synergistically decreased HA levels. While TPA increased HA levels, RA failed to increase the HA levels when compared to treatment with dimethyl sulfoxide, a solvent control. The decreasing effect of Zingiber cassumunar on HA levels was due to complete inhibiton of expression of both HAS-2 and HAS-3 mRNA after fibroblasts were treated with Zingiber cassumunar for 3 h. However, only was HAS-2 mRNA transiently up-regulated by TPA at 3 h. As previously reported, HAS-1 mRNA was not expressed in human gingival fibroblasts. Conclusions: Collectively, these data indicate the inhibitory activity of Zingiber cassumunar on HA production by inhibiting HAS-2 and HAS-3 expression in human gingival fibroblasts, and its ability to reduce tissue hydration and inflammation during wound healing may be suggested. This study was supported by the Thailand Research Fund, Grant 4580028, to S.K., and the National Research Council of Thailand to P.K.