Nasopharyngeal carcinoma (NPC) is a highly metastatic tumor characterized by close association with EBV. Of the EBV-encoded products, latent membrane protein-1 (LMP-1) is thought to be the only oncoprotein playing an essential role in cell transformation as well as tumor metastasis. In this study, we tested the effect of suppressing LMP-1 by RNA interference (RNAi) on the proliferative and metastatic potentials of an EBV-positive NPC cell line, C666. We showed that stably suppressing LMP-1 by short hairpin RNA (shRNA) plasmid significantly altered cell motility, substratum adhesion, and transmembrane invasion ability. However, it has little effect on the rate of cell growth and cell cycle control. These results demonstrated the effectiveness of RNAi in suppressing LMP-1, supporting an important role of LMP-1 in NPC metastasis, and suggested a potential application of RNAi-mediated therapeutic strategy for EBV-related NPC.