Matrix metalloproteinases (MMPs) have been regarded as major critical molecules assisting tumor cells during angiogenesis and metastasis. Enhanced expression of matrix metalloproteinase-9 (MMP-9) is associated with human non-small cell lung cancer (NSCLC) invasion and metastasis. DNAzyme is a single-stranded DNA catalyst that can be engineered to bind to its complementary sequence in the target gene and cleave the mRNA. In this study, DNAzyme targeting MMP-9 was designed and synthesized. We found it strongly inhibited MMP-9 mRNA and protein expression in the NSCLC cell line A549. Inhibition of cell proliferation, adhesion, migration and invasion was also demonstrated. Thus, DNAzyme targeting MMP-9 may be a promising anti-invasion and anti-metastasis strategy for cancer gene therapy in NSCLC.