The mechanical properties of electrodeposited copper with highly-oriented nanoscale twins were investigated by micropillar compression. Uniform nanotwinned copper films with preferred twin orientations, either vertical or horizontal, were obtained by controlling the plating conditions. In addition, an ultrafine grained copper film was synthesized to be used as a reference sample. The mechanical properties were assessed by in situ SEM microcompression of micropillars fabricated with a focused ion beam. Results show that the mechanical properties are highly sensitive to the twin orientation. When compared to the ultrafine grained sample, an increase of 44% and 130% in stress at 5% offset strain was observed in quasi-static tests for vertically and horizontally aligned twins, respectively. Inversely strain rate jump microcompression testing reveals higher strain sensitivity for vertical twins. These observations are attributed to a change in deformation mechanism from dislocation pile-ups at the twin boundary for horizontal twins to dislocations threading inside the twin lamella for vertical twins.