We investigated the microstructure changes in abnormal grain growth of nanotwinned Cu (nt-Cu) upon annealing by electron backscattered diffraction (EBSD) and focused ion beam (FIB). The nt-Cu films were produced by electrodeposition on a Si wafer with a barrier/seed layer of TiW/Cu. When a 3 μm thick nt-Cu film was annealed at 350 °C for 45 min, its microstructure was found to have changed from the (111) oriented twins to the (200) oriented grains with an average grain size larger than 100 μm. When the nt-Cu film is thicker than 5 μm, the change can occur at a lower temperature of 300 °C. We observed that there is a critical abnormal grain growth temperature, below which no microstructure change can occur. We propose that it is due to nucleation limitation. After nucleation, the growth is highly anisotropic, where the lateral growth is at the least one order of magnitude faster than the normal growth. We have attempted to use JMAK theory of phase transformation to analyze the abnormal grain growth kinetics, yet we obtained very little success.