Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron loss. Evidence suggests that mitochondrial dysfunction, apoptosis, oxidative stress, inflammation, glutamate excitotoxicity, and proteasomal dysfunction are all responsible for ALS pathogenesis. N-acetyl-tryptophan has been identified as an inhibitor of mitochondrial cytochrome c release and therefore is a potential neuroprotective agent. By quantifying cell death, we demonstrate that N-acetyl-l-tryptophan (L-NAT) and N-acetyl-DL-tryptophan are neuroprotective in NSC-34 motor neuron-like cells and/or primary motor neurons, while their isomer N-acetyl-d-tryptophan has no protective effect. These findings are consistent with energy minimization and molecular modeling analysis, confirming that L-NAT generates the most stable complex with the neurokinin-1 receptor (NK-1R). L-NAT inhibits the secretion of Substance P and IL-1β (Enzyme-Linked Immunosorbent Assay and/or dot blots) and mitochondrial dysfunction by effectively inhibiting the release of cytochrome c/Smac/AIF from mitochondria into the cytoplasm and activation of apoptotic pathways, including the activation of caspase-1, -9, and -3, as well as proteasomal dysfunction through restoring chymotrypsin-like, trypsin-like, and caspase-like proteasome activity. These data provide insight into the molecular mechanisms by which L-NAT offers neuroprotection in models of ALS and suggest its potential as a novel therapeutic strategy for ALS. We demonstrate that L-NAT (N-acetyl-l-tryptophan), but not D-NAT, rescues NSC-34 cells and primary motor neurons from cell death. L-NAT inhibits the secretion of Substance P and IL-1β, and caspase-1 activation, the release of cytochrome c/Smac/AIF, and the activation of caspase -9, and -3, as well as proteasomal dysfunction. The data suggest the potential of L-NAT as a novel therapeutic strategy for amyotrophic lateral sclerosis (ALS). AIF, apoptosis-inducing factor.