Mutations in neurofilament light (NFL) subunit and small heat-shock protein B1 (HSPB1) cause autosomal-dominant axonal Charcot–Marie–Tooth disease type 2E (CMT2E) and type 2F (CMT2F). Previous studies have shown that CMT mutations in NFL and HSPB1 disrupt NF assembly and cause aggregation of NFL protein. In this study, we investigate the role of aggregation of NFL protein in the neurotoxicity of CMT mutant NFL and CMT mutant HSPB1 in motor neurons. We find that expression of CMT mutant NFL leads to progressive degeneration and loss of neuronal viability of cultured motor neurons. Degenerating motor neurons show fragmentation and loss of neuritic processes associated with disruption of NF network and aggregation of NFL protein. Co-expression of wild-type HSPB1 diminishes aggregation of CMT mutant NFL, induces reversal of CMT mutant NFL aggregates and reduces CMT mutant NFL-induced loss of motor neuron viability. Like CMT mutant NFL, expression of S135F CMT mutant HSPB1 also leads to progressive degeneration of motor neurons with disruption of NF network and aggregation of NFL protein. Further studies show that wild-type and S135F mutant HSPB1 associate with wild-type and CMT mutant NFL and that S135F mutant HSPB1 has dominant effect on disruption of NF assembly and aggregation of NFL protein. Finally, we show that deletion of NFL markedly reduces degeneration and loss of motor neuron viability induced by S135F mutant HSPB1. Together, our data support the view that disruption of NF network with aggregation of NFL is a common triggering event of motor neuron degeneration in CMT2E and CMT2F disease.