VPg (virus protein, genome-linked) is a multifunctional protein that plays important roles in viral multiplication in the cytoplasm. However, a number of VPgs encoded by plant viruses target the nucleus and this appears to be biologically significant. These VPgs may therefore be translocated between nuclear and cytoplasmic compartments during virus infection, but such nucleo-cytoplasmic transport has not been demonstrated. We report that VPg encoded by Wheat yellow mosaic virus (WYMV, genus Bymovirus, family Potyviridae) accumulated in both the nucleus and cytoplasm of infected cells, but localized exclusively in the nucleus when expressed alone in plants. Computational analyses predicted the presence of a nuclear localization signal (NLS) and a nuclear export signal (NES) in WYMV VPg. Mutational analyses showed that both the N-terminal and the NLS domains of VPg contribute to the efficiency of nuclear targeting. In vitro and in planta assays indicated that VPg interacts with WYMV coat protein (CP) and proteinase 1 (P1) proteins. Observation of VPg fused to a fluorescent protein and subcellular fractionation experiments showed that VPg was translocated to the cytoplasm when co-expressed with CP, but not with P1. Mutations in the NES domain or treatment with leptomycin B prevented VPg translocation to the cytoplasm when co-expressed with CP. Our results suggest that association with CP facilitates the nuclear export of VPg during WYMV infection.