Polysaccharides isolated from the fruiting body of Inonotus obliquus (PFIO) are known to possess various pharmacological properties including antitumor activity. However, the anti-metastatic effect and its underlying mechanistic signaling pathway involved these polysaccharides in human non-small cell lung carcinoma remain unknown. The present study therefore aimed to determine the anti-metastatic potential and signaling pathways of PFIO in the highly metastatic A549 cells. We found that PFIO suppressed the migration and invasive ability of A549 cells while decreasing the expression levels and activity of matrix metalloproteinase (MMP)-2 and MMP-9. Furthermore, PFIO decreased the phosphorylation levels of mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) as well as the expression level of COX-2, and inhibited the nuclear translocation of nuclear factor κB (NF-κB) in A549 cells. These results suggested that PFIO could suppress the invasion and migration of human lung carcinoma by reducing the expression levels and activity of MMP-2 and MMP-9 via suppression of MAPKs, PI3K/AKT, and NF-κB signaling pathways.