Epstein-Barr virus (EBV) infection is causatively related to a variety of human cancers, including nasopharyngeal carcinoma (NPC) and gastric cancer (GC). EBV encodes 44 mature miRNAs, a number of which have been proven to promote carcinogenesis by targeting host genes or self-viral genes. However, in this study, we found that an EBV-encoded microRNA, termed EBV-miR-BART6-3p, inhibited EBV-associated cancer cell migration and invasion including NPC and GC by reversing the epithelial-mesenchymal transition (EMT) process. Using microarray analysis, we identified and validated that a novel long non-coding RNA (lncRNA) LOC553103 was downregulated by EBV-miR-BART6-3p, and LOC553103 knockdown by specific siRNAs phenocopied the effect of EBV-miR-BART6-3p, while LOC553103 overexpression promoted cancer cell migration and invasion to facilitate EMT. In conclusion, we determined that EBV-miR-BART6-3p, a microRNA encoded by oncogenic EBV, inhibited EBV-associated cancer cell migration and invasion by targeting and downregulating a novel lncRNA LOC553103. Thus, our study presents an unreported mechanism underlying EBV infection in EBV-associated cancer carcinogenesis, and provides a potential novel diagnosis and treatment biomarker for NPC and other EBV-related cancers.