Previous studies in mice have shown that CD8(+) T cells can mediate clearance of rotavirus (RV) infection and provide protection from reinfection. Moreover, it has been demonstrated that VP7 can induce a cross-reactive and vigorous specific cytotoxic T-lymphocyte (CTL) response. In this study, a panel of the potential human leukocyte antigen (HLA)-A2.1-restricted CTL epitopes derived from the human RV Wa strain VP7 glycoprotein were screened and assessed using reverse immunogenetics. The specific CTLs induced in vitro by p18-26 (LLNYILKSV)-pulsed autologous dendritic cells from the peripheral blood lymphocytes of healthy HLA-A2.1 donors released interferon-gamma (IFN-gamma) specifically upon stimulation of p18-26, and meanwhile these CTLs lysed p18-26-loaded T2 cells and human RV Wa strain-infected HLA-A2.1(+) Caco-2 cells in an HLA-A2.1-restricted manner. P18-26 was still proved to be immunogenic in vivo in HLA-A2.1/Kb transgenic mice. We propose that the newly identified CTL epitope restricted by HLA-A2.1 could aid in further study of RV-associated infection in humans.