Research on the MgB 2 /Ni and MgB 2 /B multilayer films fabricated by an electron beam (EB) evaporation technique have been extensively carried out. The critical current density, J c of MgB 2 /Ni and MgB 2 /B multilayer films in parallel fields has been suggested to be higher than that of monolayer MgB 2 film due to introducing the artificial pinning centers of nano-sized Ni and B layers. Nanostructure characterization of the artificial pinning centers in the multilayer films were examined by transmission electron microscopy (TEM) and scanning TEM (STEM-energy dispersive X-ray spectroscopy (STEM-EDS))–EDS to understand the mechanism of flux pinning. The growth of columnar MgB 2 grains along the film-thickness direction was recognized in the MgB 2 /Ni multilayer film, but not in the MgB 2 /B multilayer film. Nano-sized Ni layers were present as crystalline epitaxial layers which is interpreted that Ni atoms might be incorporated into the MgB 2 lattice to form (Mg,Ni)B 2 phase. On the other hand, nano-sized B layers were amorphous layers. Crystalline (Mg,Ni)B 2 layers worked more effectively than amorphous B-layers, providing higher flux-pinning force that resulted in higher J c of the MgB 2 /Ni multilayer film than the MgB 2 /B multilayer film.