We investigated the molecular mechanisms involved in the expression of matrix metalloproteinase-9 (MMP-9) and the inhibition of MMP expression by magnolol in 5637 human urinary bladder cancer cells. Tumor necrosis factor-α (TNF-α) stimulated the secretion of MMP-9 in 5637 cells, as shown by zymography and promoter assay. The transcription factor nuclear factor kappaB (NF-κB) binding site was identified by gel-shift assay to be a cis-element for TNF-α activation of the MMP-9 promoter. Our results also demonstrated that TNF-α stimulates MMP-9 expression via the p38 MAP kinase signaling pathway in 5637 cells. Moreover, p38 MAP kinase-mediated MMP-9 gene regulation in response to TNF-α is involved in the NF-κB response element in 5637 cells. In addition, magnolol inhibited TNF-α-induced expression of the MMP-9, as determined by zymography and immunoblot, in 5637 cells. The TNF-α-induced invasion and migration of cells was inhibited by magnolol, as assessed by a modified boyden chamber and wound-healing assays, respectively. Finally, magnolol blocked MMP-9 expression, at least in part, by decreasing the binding of transcription factor NF-κB to DNA. In conclusion, TNF-α induced MMP-9 expression in 5637 cells by activating the transcription factor NF-κB, which is involved in the p38 MAP kinase-mediated control of MMP-9 regulation. Magnolol inhibited MMP-9 expression through the transcription factor NF-κB in TNF-α-induced 5637 cells.