Neurofilament light gene mutations have been linked to a subset of patients with Charcot‐Marie‐Tooth disease, the most common inherited motor and sensory neuropathy. We have previously shown that Charcot‐Marie‐Tooth‐linked mutant neurofilament light assembles abnormally in non‐neuronal cells. In this study, we have characterized the effects of expression of mutant neurofilament light proteins on axonal transport in a neuronal cell culture model. We demonstrated that the Charcot‐Marie‐Tooth‐linked neurofilament light mutations: (i) affect the axonal transport of mutant neurofilaments; (ii) have a dominant‐negative effect on the transport of wild‐type neurofilaments; (iii) affect the transport of mitochondria and the anterograde axonal transport marker human amyloid precursor protein; (iv) result in alterations of retrograde axonal transport and (v) cause fragmentation of the Golgi apparatus. Increased neuritic degeneration was observed in neuronal cells overexpressing neurofilament light mutants. Our results suggest that these generalized axonal transport defects could be responsible for the neuropathy in Charcot‐Marie‐Tooth disease.