In order to limit the scattering of electron at grain boundaries (GBs) which contributes to the resistivity, it is necessary to reduce the number of GBs by increasing the grain size of Cu films. The grain growth evolution in nanotwinned Cu (nt-Cu) films prepared by electrodeposition is investigated in this study. For the purpose of understanding the nt-Cu grain growth occur on (100)-oriented Cu, a highly-textured nt-Cu film on (100)-oriented Cu seed layer has been electroplated. After that, the nt-Cu grain growth characterization on (100)-oriented Cu seed layer was performed. As the annealing temperature increases, the grain growth is enhanced; meanwhile the (111)-oriented nt-Cu film gradually grow into (100)-oriented Cu films. Cross sectional microstructure observations demonstrate the process of grain growth on bilayer Cu. Our results show that the grain size can be as large as 74 μm after 400 °C for 1 h for a 6 μm nt-Cu film. At temperatures around 200 °C, nt-Cu shows stability characteristics instead of growing into larger grains.