In this paper, the growth behavior of twin boundary in plated copper film was thoroughly investigated using SEM and TEM to facilitate its application as under bump metallization (UBM). The bath consisting of different amounts of H2SO4 was adopted to individually study the influence of pH on cross-sectional microstructure of copper film. Increasing the sulfuric acid concentration could greatly increase the nanoscale twin formation and make it nucleate and grow from sites much nearer the wafer. Besides, nanoscale twins were only observed in columnar grains rather than equiaxed crystallites. The growth rate of copper film was found to be enhanced with acid amount, and it was found that the stacking faults rise consequently which contributed to the nucleation and extension of the nanoscale twins. The unevenly distributed acid concentration in the bath could “recrystallize” the columnar grains into equiaxed grains, and nanoscale twins were transformed into low density of thick “annealed twins”. This could illuminate why columnar grains form ahead of equiaxed grains and coherent twin boundaries nucleated in columnar grains repetitively along the growth direction. Here, the influence of sulfuric acid was first confirmed to be the essential factor in nanoscale twin formation and this may show a distinct insight into nanotwinned copper fabrication and promote its application.