To explore HER2 expression in Epstein-Barr virus-associated gastric carcinoma (EBVaGC) and the possible mechanisms causing down-regulation of HER2 expression in EBVaGC, we first evaluated HER2 and LMP2A expression on a clinicopathological-features matched cohort including 78 EBVaGC and 216 EBV-negative gastric carcinoma (EBVnGC) cases by immunohistochemistry. Cases with high HER2 expression in EBVaGC were significantly less than in EBVnGC (5.1% versus 23.7%; p<0.001), and none of the 34 LMP2A+ EBVaGC showed high HER2 expression. Further, overexpressing LMP2A in EBV-negative SGC7901 cells significantly decreased HER2, TWIST and YB-1 mRNA by 36.1%±8.1%, 87.6%±14.0% and 83.8%±5.7%, and protein by 44%, 57% and 49%, respectively. Additionally, the nucleus/cytoplasm ratios of TWIST and YB-1 were also decreased by 85% and 80%, respectively. Silencing LMP2A by siRNA in EBV-positive SNU719 cells for 48 h significantly increased HER2, TWIST and YB-1 mRNA to 276.7%±14.6%, 1284.8%±38.2% and 332.0%±15.5% and protein to 212%, 457% and 232%, respectively. The nucleus/cytoplasm ratios of TWIST and YB-1 were up-regulated by 4.00- and 3.57-fold, respectively, following LMP2A down-regulation. Moreover, LMP2A+/HER2low EBVaGC cases presented the best overall survival compared with LMP2A-/HER2low and LMP2A-/HER2high cases (p=0.003, log-rank test). These results suggest that LMP2A may suppress the HER2 expression through the TWIST/YB-1 axis in EBVaGC.