Numerous virus pathogens are transmitted by specific arthropod vectors. Understanding the mechanism of transmission is a critical step in the epidemiology of plant viruses and is crucial for the development of effective disease control strategies. In this study, we describe the localization and distribution of Wheat dwarf virus (WDV), an economically important and widespread single-stranded DNA virus, in its leafhopper vector, Psammotettix alienus. The results suggest that WDV not only can move to the salivary glands from the anterior and middle midgut via the hemocoel but also can pass directly through the sheath of the filter chamber and be readily transmitted to healthy wheat plants within 5 min of an acquisition access period on infected plants. When a bacterial-expressed recombinant capsid protein (CP) was incubated with the internal organs of leafhoppers, CP-immunoreactive antigens were found at the anterior and middle midgut. Furthermore, when leafhoppers were fed with an antiserum raised against the CP, the accumulation of WDV in the gut cells, hemocoel, and salivary glands was significantly reduced. These data provide evidence that transmission of WDV is determined by a CP-mediated virion-vector retention mechanism.