Cancer stem cells (CSCs) are cancer-initiating cells that are not only a source of tumorigenesis but also the cause of tumour progression, metastasis and therapy resistance.EBV-associated gastric cancer (EBVaGC) is a distinct subtype of gastric cancer with unique clinicopathological and molecular features. However, whetherCSCs exist inEBVaGC, and the tumorigenic mechanism ofEBV, remains unclear. Here,NOD/SCIDmice were injected subcutaneously with theEBVaGCcell lineSNU719 and treated with 5-fluorouracil weekly. Successive generations of xenografts yielded a highly malignantEBVaGCcell line,SNU-4th, which displays properties ofCSCs and mainly consists ofCD44(+)CD24(-)cells. In SNU-4th cells, an EBV-encoded circRNA, ebv-circLMP2A, expression increased and plays crucial roles in inducing and maintaining stemness phenotypes through targeting miR-3908/TRIM59/p53 axis. Additionally, high expression of ebv-circLMP2A is significantly associated with metastasis and poor prognosis in patients withEBVaGC. These findings not only provide evidence for the existence ofCSCs inEBVaGCand elucidate the pathogenic mechanism of ebv-circLMP2A inEBVaGC, but also provide a promising therapeutic target forEBVaGC.