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The softening effect of ultrasonic vibration on pure copper is studied from a new perspective with micro-tensile tests, where the gauge length of the specimen is one order of magnitude smaller than the ultrasonic wavelength. With this configuration, the amount of flow stress reduction increases linearly with vibration amplitude whereas the flow stress reduction is insensitive to the studied strain rate ranging from 0.06/s to 1/s. Temperature rise associated with ultrasonic vibration is minimal from infrared thermal imaging. In situ digital image correlation (DIC) analysis shows strain localization near ultrasonic source whereas uniform strain distribution was observed during conventional tensile test. Optical microstructure characterization shows that area fraction of annealing twins in the deformed copper reduced from 3.3% to 1.8% with ultrasonic vibration. This is possibly attributed to enhanced interaction of dislocation between twin boundaries which act as non-regenerative dislocation source. Electron backscatter diffraction (EBSD) results show that ultrasonic vibration promotes preferential grain re-orientation and reduces the misorientation within grains.