It is well established that motor neurons with large axon caliber are selectively affected in amyotrophic lateral sclerosis (ALS). To investigate whether high neurofilament (NF) content and large axonal caliber are factors that predispose motor neurons to selective degeneration in ALS, we generated mice expressing a mutant form of superoxide dismutase 1 (SOD1 G37R ) linked to familial ALS in a context of one allele for each NF gene being disrupted. A ≈40% decrease of NF protein content detected in triple heterozygous knockout mice shifted the calibers of large axons in L5 ventral root from 5–9 μm to 1–5 μm, altering neither the normal subunit stoichiometry and morphological distribution of NFs nor levels of other cytoskeletal proteins. This considerable reduction in NF burden and caliber of axons did not extend the life span of SOD1 G37R mice nor did it alleviate the loss of motor axons. Moreover, increasing the density of NFs in axons by overexpressing a NF-L transgene did not accelerate disease in SOD1 G37R mice. These results do not support the current view that high NF content and large caliber of axons may account for the selective vulnerability of motor neurons in ALS caused by mutant SOD1.