Serine palmitoyltransferase (SPT) is a key enzyme in the first step of sphingolipid biosynthesis. Mutations in theSPTLC1gene that encodes for SPT subunits cause hereditary sensory neuropathy type 1. However, little is understood about how mutant SPT regulates mechanisms of sensory neuron and axonal growth. Using transgenic mice overexpressing the C133W SPT mutant, we found that mutant dorsal root ganglia (DRG) during growthin vitroexhibit increased neurite length and branching, coinciding with elevated expression of actin-cross-linking proteins at the neuronal growth cone, namely phosphorylated Ezrin/Radixin/Moesin. In addition, inhibition of SPT was able to reverse the mutant phenotype. Because mutant SPT preferentially usesl-alanine over its canonical substratel-serine, we also investigated the effects of substrate availability on DRG neurons. Supplementation withl-serine or removal ofl-alanine independently restored normal growth patterns in mutant SPTLC1^C133WDRG. Therefore, we report that substrate availability and selectivity of SPT influence the regulation of neurite growth in DRG neurons.SIGNIFICANCE STATEMENTHereditary sensory neuropathy type 1 is an autosomal-dominant disorder that leads to a sensory neuropathy due to mutations in the serine palmitoyltransferase (SPT) enzyme. We investigated how mutant SPT and substrate levels regulate neurite growth. Because SPT is an important enzyme in the synthesis of sphingolipids, our data are of broader significance to other peripheral and metabolic disorders.