Matrix metalloproteinase-2 (MMP-2) has pivotal role in the degradation of extracellular matrix, and thereby enhances the invasive, proliferative and metastatic potential in cancer. Knockdown of MMP-2 using MMP-2 small interfering RNA (pM) in human glioma xenograft cell lines 4910 and 5310 decreased cell proliferation compared with mock and pSV (scrambled vector) treatments, as determined by 5-bromo-2'-deoxyuridine incorporation, Ki-67 staining and clonogenic survival assay. Cytokine array and western blotting using tumor-conditioned media displayed modulated secretory levels of various cytokines including granulocyte-macrophage colony-stimulating factor, interleukin-6 (IL-6), IL-8, IL-10, tumor necrosis factor-α, angiogenin, vascular endothelial growth factor and PDGF-BB in MMP-2 knockdown cells. Further, cDNA PCR array indicated potential negative regulation of Janus kinase/Stat3 pathway in pM-treated cells. Mechanistically, MMP-2 is involved in complex formation with α5 and β1 integrins and MMP-2 downregulation inhibited α5β1 integrin-mediated Stat3 phosphorylation and nuclear translocation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays showed inhibited Stat3 DNA-binding activity and recruitment at CyclinD1 and c-Myc promoters in pM-treated cells. In individual experiments, IL-6 or siRNA-insensitive MMP-2 overexpression by pM-FL-A141G counteracted and restored the pM-inhibited Stat3 DNA-binding activity, suggesting IL-6/Stat3 signaling suppression in pM-treated 4910 and 5310 cells. MMP-2/α5β1 binding is enhanced in human recombinant MMP-2 treatments, resulting in elevated Stat3 DNA-binding activity and recruitment on CyclinD1 and c-Myc promoters. Activation of α5β1 signaling by Fibronectin adhesion elevated pM-inhibited Stat3 phosphorylation whereas blocking α5β1 abrogated constitutive Stat3 activation. In vivo experiments with orthotropic tumor model revealed the decreased tumor size in pM treatment compared with mock or pSV treatments. Immunofluorescence studies in tumor sections corroborated our in vitro findings evidencing high expression and co-localization of MMP-2/α5β1, which is decreased upon pM treatment along with significantly reduced IL-6, phospho-Stat3, CyclinD1, c-Myc, Ki-67 and PCNA expression levels. Our data indicate the possible role of MMP-2/α5β1 interaction in the regulation of α5β1-mediated IL-6/Stat3 signaling activation and signifies the therapeutic potential of blocking MMP-2/α5β1 interaction in glioma treatment.