Glycoprotein B (gB) is an essential fusion protein for Epstein-Barr virus (EBV) infection of both B cells and epithelial cells and is thus a promising target antigen for a prophylactic vaccine to prevent or reduce EBV-associated disease. T cell responses play key roles in the control of persistent EBV infection and the efficacy of a vaccine. However, to date, T cell responses to gB have been characterized for only a limited number of human leukocyte antigen (HLA) alleles. Here, we screened gB T cell epitopes in 23 healthy EBV carriers and 10 patients with nasopharyngeal cancer (NPC) using a peptide library spanning the entire gB sequence. We identified 12 novel epitopes in the context of seven new HLA restrictions that are common in Asian populations. Two epitopes, gB(214-223) and gB(840-849), restricted by HLA-B*58:01 and -B*38:02, respectively, elicited specific CD8(+) T cell responses to inhibit EBV-driven B cell transformation. Interestingly, gB-specific CD8(+) T cells were more frequent in healthy viral carriers with EBV reactivation than in those without EBV reactivation, indicating that EBV reactivation in vivo stimulates both humoral (VCA-gp125-IgA) and cellular responses to gB. We further found that most gB epitopes are conserved among different EBV strains. Our study broadens the diversity and HLA restrictions of gB epitopes and suggests that gB is a common target of T cell responses in healthy viral carriers with EBV reactivation. In particular, the precisely mapped and conserved gB epitopes provide valuable information for prophylactic vaccine development. IMPORTANCE T cells are crucial for the control of persistent EBV infection and the development of EBV-associated diseases. The EBV gB protein is essential for virus entry into B cells and epithelial cells and is thus a target antigen for vaccine development. Understanding T cell responses to gB is important for subunit vaccine design. Here, we comprehensively characterized T cell responses to full-length gB. Our results expand the available gB epitopes and HLA restrictions, particularly those common in Asian populations. Furthermore, we showed that gB-specific CD8(+) T cells inhibit B cell transformation ex vivo and that gB-specific CD8(+) T cell responses in vivo may be associated with intermittent EBV reactivation in asymptomatic viral carriers. These gB epitopes are highly conserved among geographically separated EBV strains. Precisely mapped and conserved T cell epitopes may contribute to immune monitoring and the development of a gB subunit vaccine.