Cellulose, a major constituent of our natural environment and a structured biodegradable biopolymer, has been shown to exhibit shear piezoelectricity with potential applications in energy harvesters, biomedical sensors, electro-active displays and actuators. In this regard, a high-aspect ratio nanofiber geometry is particularly attractive as flexing or bending will likely produce a larger piezoelectric response as compared to axial deformation in this material. Here we report self-assembled cellulose nanofibers (SA-CNFs) fabricated using a template-wetting process, whereby parent cellulose nanocrystals (CNCs) introduced into a nanoporous template assemble to form rod-like cellulose clusters, which then assemble into SA-CNFs. Annealed SA-CNFs were found to exhibit an anisotropic shear piezoelectric response as directly measured using non-destructive piezo-response force microscopy (ND-PFM). We interpret these results in light of the distinct hierarchical structure in our template-grown SA-CNFs as revealed by scanning electron microscopy (SEM) and high resolution transmission electron microscopy (TEM).