Rep78/68 proteins of adeno-associated virus type 2 (AAV-2) are involved in many aspects of the viral life cycle, including replication, gene expression, and site-specific integration. To understand the molecular mechanisms of the actions of Rep proteins, we searched for Rep68-interacting cellular proteins by utilizing a one-step affinity purification technique and identified two members of 14-3-3 proteins (14-3-3 epsilon and gamma). We found that phosphorylation of 535Ser at the carboxy terminus of Rep68 was critical for its association with 14-3-3. The association of 14-3-3 proteins to Rep68 resulted in reduction of the affinity of Rep68 for DNA. Furthermore, genome DNA replication of a recombinant mutant virus carrying a phosphorylation-deficient Rep68 (Ser535Ala) was more efficient than that of the wild-type virus. These results suggest that phosphorylation of Rep68 and subsequent association with 14-3-3 proteins regulates Rep-mediated functions during the AAV life cycle.