Tumor necrosis factor (TNF)-alpha is one of the pro-inflammatory cytokines highly expressed in Helicobacter pylori that inhibits gastric acid secretion. In this study we determined the effect of silibinin on TNF-alpha-induced MMP-9 expression in gastric cancer cell lines. MMP-9 mRNA and protein expression was dose-dependently increased by TNF-alpha in SNU216 and SNU668 gastric cancer cells. On the other hand, TNF-alpha-induced MMP-9 expression was dose-dependently suppressed by silibinin. To verify the regulatory mechanism of silibinin on TNF-alpha-induced MMP-9 expression, the gastric cancer cell lines were pretreated with silibinin prior to TNF-alpha. TNF-alpha-induced MMP-9 expression was inhibited by the MEK1/2 specific inhibitor, UO126. Finally, we investigated the effect of adenoviral constitutively active (CA)-MEK and CA-Akt on MMP-9 expression. The expression of MMP-9 was significantly increased by CA-MEK overexpression, but not by CA-Akt overexpression. Taken together, we suggest that silibinin down-regulates TNF-alpha- induced MMP-9 expression through inhibition of the MEK/ERK pathway in gastric cancer cells.