Using Lucifer Yellow we have demonstrated that the phloem-loading pathway from the mesophyll to the bundle sheath—vascular parenchyma interface inZea mays source leaves follows a symplasmic route in small and intermediate vascular bundles in control as well as in the green sections of mutant sucrose-export-defective (SXD-1) plants. In the anthocyanin-rich mutant leaf sections, Lucifer Yellow transport was prohibited along the same path, at the bundle sheath—vascular parenchyma interface in particular. Plasmodesmata at the latter interface in SXD-1 anthocyanin-rich leaf sections appear to be structurally altered through callose deposition at the plasmodesmal orifices. We suggest that a transport bottleneck at the bundle sheath—vascular parenchyma interface is thus orchestrated and regulated through callose formation, preventing symplasmic transport across this important loading interface.