We present a method to adjust the stiffness of individual piezoelectric nanofiber by electron beam induced polarization under an in situ scanning electron microscopy. The lead zirconate titanate (PZT) nanofibers were fabricated by an electrospinning process. The Young's modulus was calculated from the resonant frequency excited by an oscillating electric field applied through a nanomanipulator. The stiffness can be adjusted up to 75% by induced polarization under the exposure of an electron beam to control the domain boundaries in single PZT nanofibers. Splitting effect of the resonant frequencies was observed due to anisotropic stiffness in polarized PZT nanofibers. (C) 2011 American Institute of Physics. [doi: 10.1063/1.3660226]