MMP-9 participates in tumour growth, invasion, metastasis and vascularisation. Thus, inhibition of MMP-9 may be involved in the process of tumourigenesis. We have investigated the effect of RNAi-mediated MMP-9 silencing on inhibiting invasion and migration of mouse melanoma cell B16. A specific and optimised siRNA vector was used to target MMP-9 mRNA synthesis in B16 cells. Changes of invasion and migration capability of B16 cell were examined after transfection at different time, and a footpad tumour model was performed to measure the effect of MMP-9 siRNA on melanoma tumourigenesis in vivo. In vitro, down-regulation of MMP-9 expression significantly inhibited B16 cell invasion and migration. In vivo, intratumoural injection of plasmid DNA expressing MMP-9 siRNA every 3 days for three times remarkably inhibited melanoma growth and also suppressed tumour metastasis. The results indicate that RNA-mediated targeting of MMP-9 may have promising applications for the treatment of melanoma.