The distribution of Rhenium (Re) in a Ni-based single-crystal superalloy is studied by sub-angstrom resolution transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDS). It is found that Re atoms segregate at the tensile stress regions near the interfacial dislocation cores, forming the “Cottrell atmosphere”, and the segregation process is facilitated by dislocation pipe diffusion. In situ TEM and scanning electron microscopy (SEM) straining studies reveal that the Re-decorated dislocation networks along the phase boundaries act as mechanical walls that effectively block dislocation motion and crack propagation. Furthermore, the degree of Re segregation can be regulated by thermal treatment. Theoretical analysis demonstrates that this remarkable alloying effect originated mainly from the interactions between the local composition strain of Re and the dislocation strains, leading to significantly stabilized interfacial dislocation networks. These results provide a new perspective on understanding the origin of the Re effect on mechanical properties in Ni-based superalloys and will be beneficial to both improving creep properties of Ni-based superalloys and designing high-performance Re-free superalloys. Display omitted