Alsin gene mutations were found to cause autosomal recessively inherited rare form of juvenile ALS (amyotrophic lateral sclerosis 2/ALS2). The loss of function in the alsin protein results in selective and progressive motor neuron degeneration. The identification of alsin as the cause of two other childhood motor neuron diseases—juvenile primary lateral sclerosis and infantile-onset hereditary spastic paraplegia—suggests that alsin plays a critical role in motor neuron survival. Alsin protein is known to have activation domains for three members of Ras superfamily guanosine triphosphatases, and might be involving in several key pathways including development, proliferation, and differentiation. Despite extensive research, the mechanisms underlying ALS2-mediated neurodegeneration remain poorly understood, partly due to the inability of animal models to fully replicate the disease phenotype and the lack of patient-specific disease models. In this study, we have investigated alsin related molecular pathophysiology in an ALS2 patient with a homozygous c.3415C>T (p.Arg1139Ter) mutation in both fibroblasts and iPSC-derived cortical motor neuron-like cells (iNeurons). The results were confirmed in ALS2 knockdown iNeurons generated via CRISPR interference. The findings suggested that GEF activity of alsin for Rac1-GTPase and endosomal trafficking were impaired, and these pathways might be contributing to the disease pathogenesis in this patient. Future studies in patients with different ALS2 mutations will be crucial to unraveling the shared molecular mechanisms of ALS2-related disorders. A thorough understanding of the biological functions of alsin and the molecular pathophysiology resulting from its loss is essential for identifying new therapeutic targets and establishing molecular biomarkers for treatment.