Many herpesviral immediate-early proteins promote their robust lytic phase replications by hijacking the cell cycle machinery. Previously, lytic replication of Epstein-Barr virus (EBV) was found to be concurrent with host cell cycle arrest. In this study, we showed that ectopic expression of EBV immediate-early protein Rta in HEp-2 cells resulted in increased G1/S population, hypophosphorylation of pRb and decreased incorporation of 5-bromo-2'-deoxyuridine. In addition, EBV Rta transcriptionally upregulates the expressions of p21 and 14-3-3σ in HEp-2 cells, 293 cells and nasopharyngeal carcinoma TW01 cells. Although p21 and 14-3-3σ are known targets for p53, Rta-mediated p21 and 14-3-3σ transactivation can be detected in the absence of p53. In addition, results from luciferase reporter assays indicated that direct binding of Rta to either promoter sequences is not required for activation. On the other hand, a special class of Sp1-responsive elements was involved in Rta-mediated transcriptional activation on both promoters. Finally, Rta-induced p21 expression diminished the activity of CDK2/cyclin E complex, and, Rta-induced 14-3-3σ expression sequestered CDK1 and CDK2 in the cytoplasm. Based on these results, we hypothesize that through the disruption of CDK1 and CDK2 activities, EBV Rta might contribute to cell cycle arrest in EBV-infected epithelial cells during viral reactivation.