The peel strength of a copper (Cu) thin film deposited on a polyimide (PI) substrate deteriorates after heat treatment at 150 °C in air. The deterioration involves Cu2O microparticles (10–100 nm) penetrating the PI substrate, both the substrate and film being oxidized. To prevent this penetration and oxidation, we introduced an interfacial layer (vanadium, titanium, or cobalt) at the interface between the Cu thin film and PI substrate. Both titanium and cobalt interfacial layers were effective as barriers against the penetration of Cu2O particles. The quality of the interfacial layers (e.g., quantity of dislocations) influenced their effectiveness. By annealing the cobalt interfacial layers at 280 °C for 1 h in a vacuum before sputter deposition of a Cu thin film, we succeeded in keeping the peel strength between the Cu thin film and the PI substrate above 0.1 N/m even after heat treatment of the laminate at 150 °C for three days.