Passive-dynamic ankle-foot orthosis (PD-AFO) bending stiffness is a key functional characteristic for achieving enhanced gait function. However, current orthosis customization methods inhibit objective premanufacture tuning of the PD-AFO bending stiffness, making optimization of orthosis function challenging. We have developed a novel virtual functional prototyping (VFP) process, which harnesses the strengths of computer aided design (CAD) model parameterization and finite element analysis, to quantitatively tune and predict the functional characteristics of a PD-AFO, which is rapidly manufactured via fused deposition modeling (FDM). The purpose of this study was to assess the VFP process for PD-AFO bending stiffness. A PD-AFO CAD model was customized for a healthy subject and tuned to four bending stiffness values via VFP. Two sets of each tuned model were fabricated via FDM using medical-grade polycarbonate (PC-ISO). Dimensional accuracy of the fabricated orthoses was excellent (average 0.51 ± 0.39 mm). Manufacturing precision ranged from 0.0 to 0.74 Nm/deg (average 0.30 ± 0.36 Nm/deg). Bending stiffness prediction accuracy was within 1 Nm/deg using the manufacturer provided PC-ISO elastic modulus (average 0.48 ± 0.35 Nm/deg). Using an experimentally derived PC-ISO elastic modulus improved the optimized bending stiffness prediction accuracy (average 0.29 ± 0.57 Nm/deg). Robustness of the derived modulus was tested by carrying out the VFP process for a disparate subject, tuning the PD-AFO model to five bending stiffness values. For this disparate subject, bending stiffness prediction accuracy was strong (average 0.20 ± 0.14 Nm/deg). Overall, the VFP process had excellent dimensional accuracy, good manufacturing precision, and strong prediction accuracy with the derived modulus. Implementing VFP as part of our PD-AFO customization and manufacturing framework, which also includes fit customization, provides a novel and powerful method to predictably tune and precisely manufacture orthoses with objectively customized fit and functional characteristics.