Cu is effective on the refinement of Fe3O4 rust particles but its mechanism is not clarified. In this study, the influence of Cu on the nanostructure of Fe3O4 particles was investigated through a detailed analysis of the characteristics of the artificially synthesized Fe3O4 particles formed with Cu2+ by various means. X‐ray absorption fine structure analysis showed that Cu is substituted for octahedral Fe and exists as CuxFe(3‐x)O4 in Fe3O4. The first‐principle calculation showed that lattice strain resulted around the substituted Cu. These results suggest that this lattice strain around substituted Cu inhibited the lattice growth and contributed to the refinement of Fe3O4 particles.