Matrix metalloproteinase 9 (MMP-9) has been implicated in airway injury in chronic obstructive pulmonary disease (COPD), lung inflammation, and lung cancer and plays a major role in tumor necrosis factor-α (TNF-α)-stimulated tumor invasion and lung inflammation. MMP-9 activity is promoted by the pro-inflammatory cytokine TNF-α. GMI, cloned from Ganoderma microsporum and purified, is one of the recombinant fungal immunomodulatory proteins. To understand the molecular mechanisms involved in the suppression of TNF-α-mediated tumor invasion and inflammation, GMI modulation of this pathway was investigated in human alveolar epithelial A549 cells in this study. GMI exhibited an inhibitory effect on TNF-α-induced invasion, with GMI treatment and TNF-α exposure presenting the most anti-invasive properties on Boyden chamber assay. GMI reduced TNF-α-induced MMP-9 activities on gelatin zymography assay through inhibition of MMP-9 transcriptional activity. RT-PCR and MMP-9 promoter luciferase analysis revealed that GMI inhibits the transcription of MMP-9 mRNA. Moreover, in vitro and in vivo binding experiments, an electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation assay (ChIP) demonstrated that GMI suppresses DNA binding of nuclear factor (NF)-κB transcription factors to MMP-9 promoter. Western blot analysis indicated that GMI blocks the phosphorylation and degradation of IκBα, which in turn leads to suppression of the phosphorylation and nuclear translocation of p65. Thus, overall, our results indicated that GMI mediates antitumor invasion and anti-inflammatory effects through modulation of NF-κB/MMP-9 pathways.