Background. Epstein-Barr virus (EBV) infection elicit polyfunctional CD8+ T cell response. Follicular CD8+ T cells with high C-X-C chemokine receptor type 5 (CXCR5) expression have antiviral effects in chronic viral infection. However, the role of follicular CXCR5+CD8+ T cells during primary EBV infection remains unclear. Methods. Using multicolor flow cytometry, we investigated the dynamic changes of circulating follicular CXCR5+CD8+ T cells in EBV-infected infectious mononucleosis (IM) patients. Evaluation of CXCR5+CD8+ response to EBV peptides was performed. EBV DNA viral loads in PBMC were also determined. Results. Circulating follicular CXCR5+CD8+ T cells emerged gradually but EBV DNA viral loads steadily decreased over time after EBV infection. Importantly, the upregulation of CXCR5+CD8+ T cells was correlated with EBV DNA reduction. Programmed cell death 1 (PD-1) on CXCR5+CD8+ T cells emerged over time. Furthermore, CXCR5+CD8+ T cells were demonstrated to be polyfunctionality (IFN-γ and IL-2 secretion) with enhanced cytotoxicity to EBV peptides. Conclusions. Polyfunctional CXCR5+CD8+ T cells emerge during persistent EBV infection.