Effects of the Epstein-Barr virus (EBV) on cellular protein expression are essential for viral pathogenesis. Tocharacterize the cellular response to EBV infection, differential proteomes of gastric epithelial AGS cellswereanalyzed with two-dimensional gel electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionizationtimeof flight (MALDI-TOF) and liquid chromatography electrospray/ionization ion trap (LC–ESI–IT) massspectrometry identification. Mass spectrometry identified 9 altered cellular proteins, including 5 up-regulatedand 4 down-regulated proteins after EBV infection. Notably 2-DE analysis revealed that EBV infection inducedincreased expression of heat shock cognate 71 kDa protein, actin cytoplasmic 1, pyridoxine-5’-phosphate oxidase,caspase 9, and t-complex protein 1 subunit alpha. In addition, EBV infection considerably suppressed thosecellular proteins of zinc finger protein 2, cyclin-dependent kinase 2, macrophage-capping protein, and growth/differentiation factor 11. Furthermore, the differential expressional levels of partial proteins (cyclin-dependentkinase 2 and caspase 9) were confirmed by Western blot analysis.Thus, this work effectively provided usefulprotein-related information to facilitate further investigation of the mechanisms underlying EBV infection andpathogenesis.