Nanotwinned (NT) metals are promising structural materials due to their excellent combination of strength and ductility. These superior properties are strongly dependent on the microstructures i.e. the twin length (grain size), the twin thickness and the twin orientation. Understanding the synthesis process and growth mechanism of NT metals is essential for their structure design. In this work, the effect of electrolyte temperature on the microstructures of highly oriented NT Cu samples, including twin thickness and twin length (grain size), are systematically studied. The NT Cu samples were prepared by means of the direct-current electrodeposition at 293, 298, 303, 308 and 313 K, respectively, while other deposition parameters such as current density, concentration of additive and pH value were kept constant. With decreasing the temperature from 313 K to 293 K, the average grain size decreases from 27.6 μm to 2.8 μm and the average twin thickness decreases from 111 nm to 28 nm, which results in an increment of hardness from 0.7 GPa to 1.5 GPa. This is because with decreasing the temperature, the overpotential of cathode for depositing metal elevates, leading to the nucleation rate of both the grain and twin enhanced.