Charcot-Marie-Tooth Type1A (CMT1A) is a common inherited human neurological disorder that affects the peripheral nerves thereby causing weakness and atrophy of muscles. Mutations in the peripheral myelin protein PMP22 are associated with CMT1A. PMP22 is a short-lived 22 kDa glycoprotein, which is highly expressed in Schwann cells. It plays a key role in the maintenance of myelin structure and compaction. PMP22 forms aggregates when the proteasome is inhibited or the protein is mutated. We hypothesize that PMP22 aggregates and mutations lead to structural and mechanical alterations in the affected nerve fibers. To test this hypothesis, we apply the powerful nano-approach atomic force microscopy (AFM). Using AFM and the common CMT1A model Trembler-J (Tr-J) mouse, an approach has been established which enables structural and mechanical investigation at the nano-scale on living isolated nerve fibers. AFM images show significant topographical differences in the extracellular matrix and basal lamina organization between control and Tr-J/+ nerve fibers. Immunocytochemical analysis indicates that PMP22 relocates together with type IV collagen (a basal lamina ubiquitous component) to the perinuclear region of Tr-J/+ Schwann cells. AFM-based nano-indentation measurements reveal impairment in the mechanical properties of single myelinating Tr-J/+ nerve fibers. Impairment is linked to F-actin cytoskeleton re-organization in the nerve fibers. In conclusion, our observations provide valuable insight into understanding the pathophysiology of CMT1A. Furthermore, the approach used may be extended to investigating other neuropathies affecting nerve fibers.