Matrix metalloproteinase 26 (MMP-26) is a novel member of the matrix metalloproteinase (MMP) family and is widely expressed in cancer cells of epithelial origin. MMP-26 has been shown to contribute to tumor development and to the restoration of tissue injury. In this study, in order to identify the functions of MMP-26 that contribute to the biological phenotype and behavior of human lung carcinoma A549 cells, we established an MMP-26 low-expressing tumor cell model using RNA interference (RNAi) transfection. These cells were used to investigate the role of MMP-26 in tumor progression. The MTT, colony forming, adhere-nce and spreading, wound-healing and Transwell chamber invasion assays were performed to analyze the invasion ability of pshRNA-MMP26-transfected A549 cells. Semi-quantitative reverse transcription polymerase chain reaction, Western blotting and double immunofluorescent staining were employed to detect the relationship between MMP-26 and MMP-9. Results showed that the adhesive rate was down-regulated in pshRNA-MMP26-transfected cells, compared to the controls. Silencing of the MMP-26 gene significantly retarded the invasiveness of A549 cells in Transwell insert invasion assays. The cells proliferated in the three-dimensional (3D) culture system. A549 cells transfected with the pshRNA-MMP26-C plasmid mainly developed reticular structures in morphology, and formed few clones with clear and smooth edges as well as tight intercellular junctions. The mRNA and protein expression of MMP-9 in pshRNA-MMP26-transfected cells were significantly lower than those of the controls. Double immunofluorescence labeling and focal laser scanning microscopy showed that MMP-26 was colocalized with MMP-9 in the controls. In conclusion, we successfully established an MMP-26 low-expressing cell model and confirmed that MMP-26 contributed to A549 cell invasion and migration in vitro. We also demonstrated that MMP-26 plays an important role in local invasion at least in part through coordination with MMP-9.