The purpose of this study was to explore the inhibiting role of MMP-9 gene silence in the invasive ability and growth of laryngeal squamous cell carcinoma (LSCC) by lentivirus mediated RNA interference. MMP-9-RNAi-lentivirus and the control lentivirus (GFP-lentivirus) were transfected into Hep-2 cells. Gelatin zymography showed the proteins expression of MMP-9 were knockdown in the MMP-9 siRNA transfected Hep-2 cells. The invasive activity and viability of MMP-9 siRNA treated Hep-2 cells were decreased than the control cells measured with modified Boyden chamber assay and MTT assay. In animal experiment, 20 nude mice bearing Hep-2 cell tumor were randomly separated into the experimental and the control groups. The former were intratumorally injected with MMP-9-RNAi-lentivirus, and the later were injected with equivalent dose of GFP-lentivirus. Results showed the average weight and volume of tumor in MMP-9-RNAi-lentivirus treated group were significantly lower than those in the control group (P < .01). The protein expressions of MMP-9 were downregulated in tumors of MMP-9-RNAi-lentivirus treatment. The PCNA index was obviously lower in the tumors of treated group than that in the control group (P < .01). These results suggest that MMP-9 gene silence by lentivirus mediated RNA interference can inhibit invasion and growth of LSCC.