Many spectacular nanotwinned Cu (nt-Cu) properties depend on the spacing of adjacent twin boundaries (TBs) inside crystal grains. The average TB spacing is typically 10-100 nm in electrodeposited nt-Cu films. This study employed template-assisted electrodeposition to grow nt-Cu nanowires with high-density TBs (average TB spacing <5 nm). The effects of Cu seed annealing and template pore size on TB spacing were systematically investigated through microstructural characterization and electrochemical analysis. A model based on the stress accumulation level and electrochemical parameters was proposed to assess the twinning criteria during the electrodeposition of nt-Cu nanowires. This research provides mechanistic insights into the nanotwinning process and highlights the potential for the control of twin structures in nanomaterials.