Interdiffusion between sputter-deposited thin Y–Ba–Cu–O films and MgO (100) substrates has been reinvestigated by applying 3.2 Mev 4He+ Rutherford backscattering spectrometry (RBS) combined with scanning tunneling microscopy. The effects on the RBS spectra from large morphology variation up to “lake-like” structure of the films during annealing have been quantitatively evaluated and excluded. The obtained value of the interdiffusion coefficient of Cu in MgO is 1.2×10−21 m2 s−1, which is smaller by three orders of magnitude than the value reported by other workers.