Background Extracellular matrix (ECM) is a basic step of tumor invasion and metastasis. Matrix metalloproteinase (MMPs) family is a kind of zinc-ion-dependent endopeptidase, which can degrade almost all protein components in the extracellular matrix, destroy the histological barrier of tumor cell invasion, and play a key role in tumor invasion and metastasis. The role of MMP-9 in tumor invasion and metastasis has attracted increasing attention and is considered as the main proteolytic enzyme in this process. The aim of this study was to investigate the role of MMP-9 in human oral squamous cell carcinoma cells. Methods Human oral squamous cell carcinoma (OSCC) cells CAL27 and SCC-15 were cultured in DMEM high glucose medium with 10% FBS. The MMP-9-shRNA Lentiviral clone and control virus was constructed and transfected into OSCC cells. Cell proliferation was detected by MTT assay. Colony formation was evaluated by colony formation assay. Cell migration and invasion was evaluated using the scratch assay and transwell invasion assay. OSCC cells Knockdown with MMP-9-shRNA were injected into zebra fish and OSCC tumor model in zebra fish was established and evaluated. Results MMP-9 expression was down regulated significantly in RNA and protein level after transfection. Knockdown of MMP-9 gene by shRNA could inhibit oral squamous cell cancer cells growth and colony formation, and markedly suppress cell migration and invasion in vitro. And also knockdown of MMP-9 gene could significantly inhibit OSCC cells metastasis in zebra fish. Conclusions MMP-9 plays an important role in OSCC cells metastasis. Legal entity responsible for the study Beijing Stomatological Hospital & School of Stomatology, Capital Medical University. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.