Although many distinct mutations in a variety of genes are known to cause Amyotrophic Lateral Sclerosis (ALS), it remains poorly understood how they selectively impact motor neuron biology and whether they converge on common pathways to cause neuronal degeneration. Here, we have combined reprogramming and stem cell differentiation approaches with genome engineering and RNA sequencing to define the transcriptional and functional changes that are induced in human motor neurons by mutant SOD1. Mutant SOD1 protein induced a transcriptional signature indicative of increased oxidative stress, reduced mitochondrial function, altered subcellular transport, and activation of the ER stress and unfolded protein response pathways. Functional studies demonstrated that these pathways were perturbed in a manner dependent on the SOD1 mutation. Finally, interrogation of stem-cell-derived motor neurons produced from ALS patients harboring a repeat expansion in C9orf72 indicates that at least a subset of these changes are more broadly conserved in ALS. Display omitted •iPSC-derived motor neurons harboring SOD1 mutations exhibit cell survival deficits•Genetic correction rescues ALS-related phenotypes•RNA-seq reveals expression changes and mitochondrial and ER stress disturbances•Motor neurons exhibit inherent ER stress linked to electrical activity Motor neurons differentiated from human-ALS-patient-derived iPSCs were used to define transcriptional and functional changes arising from SOD1 mutations, which could be reversed by genome engineering of the SOD1 locus.