The development of macroscopic nanofiber orientation and microscopic crystallite and molecular chain orientation have been investigated during uniaxial stretching of electrospun poly(vinyl alcohol) (PVA) non-woven nanofiber mats. Scanning electron microscopy and stress-strain/small-angle X-ray scattering show that the macroscopic nanofiber orientation significantly increases during the initial stage of deformation, and approaches a plateau on the way of stretching. Detailed analyses of the stress-strain/wide-angle X-ray diffraction measurement and polarized Fourier transform infrared spectroscopy indicate that the microscopic crystallite and molecular chain orientation rapidly increase at the initial stage of stretching due to macroscopic nanofiber orientation. At higher deformation, the microscopic modes of orientation continuously develop as a result of the nanofiber stretching. The complicated deformation process of non-woven nanofiber mats is discussed in terms of macroscopic nanofiber orientation and the microscopic crystallite and molecular chain orientation. Display omitted