In this paper, the effect of annealing temperature (T) on the ductility of 50 nm thick nanocrystalline (NC) Cu films adhered to flexible substrates was investigated by a uniaxial tension test. It was found that the ductility and the fracture toughness (Gc) can be significantly improved through an annealing treatment. The crack onset strain of the 300 °C annealed Cu film is 18.1%, which is about twice that of the as-deposited NC Cu film. In addition, Gc of the 300 °C annealed Cu film is 1833 J m−2, which is nearly three times that of the as-deposited NC Cu film. Focused ion beam results indicate that the as-deposited film fractures with delamination and strain localization coevolving, while the as-annealed film fractures by adhering well to the substrate. At a higher T, the tensile residual stress is lower, the microstructure is more stable, and a diffusion or compound interface is generated, resulting in a better bonding between the film and the substrate. In this case, the strain localization is suppressed more effectively, causing improved ductility and Gc. Whether the film is as-deposited or as-annealed, the saturated crack spacing is about 1.41 µm, which accords well with the theoretical analysis. Intergranular fracture is suggested to be the main fracture mechanism.