The molecular mechanism underlying activation of MMP9 in non-small cell lung cancer (NSCLC) cells, which controls cancer invasiveness and metastasis, remains elusive. Here, we reported significant decrease in miR-129 and significant increases in phosphorylated EGFR and MMP9 in the resected NSCLC from the patients, compared with adjacent normal tissue. Moreover, strong correlations were detected among these three factors, the relationship of which was examined in two human NSCLC lines, A549 and H460. We found that EGF-induced EGFR phosphorylation in A549 or H460 cells activated MMP9 and, consequently, cancer invasiveness. The EGF-induced activation of MMP9 was efficiently inhibited either by an EGFR inhibitor or by an Akt inhibitor. However, miR-129 level was not affected by EGF stimulation. In addition, overexpression of miR-129 antagonized EGF-induced MMP9 activation without affecting EGFR phosphorylation in A549 or H460 cells. Taken together, our data suggest that miR-129 inhibits EGFR signaling through PI3K signal transduction cascades to regulate MMP9 expression in NSCLC. Thus, miR-129, EGFR, and MMP9 appear to be promising therapeutic targets for preventing the metastasis of NSCLC.