The nanoscale mechanical behavior of Zn0.975V0.025O (V-ZnO) piezoelectric nanofibers by electrospinning was investigated using a nanoindenter in detail. After being calcined at 700 °C, V-ZnO nanofibers are of hexagonal wurtzite phase crystal structure, and the diameter and length are in the range of 50–300 nm and several tens to several hundreds of micrometers. The statistical average values of reduced modulus and hardness are 58.7±4.2 and 3.3±0.2 GPa for the nanofibers, and they decrease by 47.2% and 34.0% in comparison with those of bulk ZnO. It indicates that size effect of the mechanical behavior was obviously observed for the nanofibers, and the mechanism is discussed in conjunction with their high surface-to-volume ratio. Indentation depth-dependent reduced modulus and hardness properties were observed at indentation depth less than 18 nm, and it is attributed to the strain gradient effect during nanoindentation.