Intronic expansion of a hexanucleotideGGGGCCrepeat in the chromosome9 open reading frame72 (C9ORF72) geneisthemajorcauseoffamilialamyotrophic lateral sclerosis(ALS)andfrontotemporaldementia.However,the cellular function of theC9ORF72 protein remains unknown. Here,wedemonstrate thatC9ORF72 regulates endosomal trafficking. C9ORF72 colocalized with Rab proteins implicated in autophagy and endocytic transport: Rab1, Rab5, Rab7 and Rab11 in neuronal cell lines, primary cortical neurons and human spinal cord motor neurons, consistent with previous predictionsthatC9ORF72 bearsRabguanine exchange factor activity.Consistent with this notion, C9ORF72 was present in the extracellular space and as cytoplasmic vesicles. Depletion of C9ORF72 using siRNA inhibited transport of Shiga toxin fromthe plasmamembrane to Golgi apparatus, internalizationofTrkBreceptorandalteredthe ratio ofautophagosomemarker lightchain3(LC3) II:LC3I, indicatingthat C9ORF72 regulates endocytosis and autophagy. C9ORF72 also colocalized with ubiquilin-2 and LC3-positive vesicles, and co-migrated with lysosome-stained vesicles in neuronal cell lines, providing further evidence thatC9ORF72regulates autophagy. Investigationof proteinsinteractingwithC9ORF72usingmassspectrometry identified other proteins implicated in ALS; ubiquilin-2 and heterogeneous nuclear ribonucleoproteins, hnRNPA2/B1 and hnRNPA1, and actin. Treatment of cells overexpressing C9ORF72 with proteasome inhibitors induced the formation of stress granules positive for hnRNPA1 and hnRNPA2/B1. Immunohistochemistry of C9ORF72 ALS patient motor neurons revealed increased colocalization between C9ORF72 and Rab7 and Rab11 compared with controls, suggesting possible dysregulation of trafficking in patients bearing the C9ORF72 repeat expansion. Hence, this study identifies a role for C9ORF72 in Rab-mediated cellular trafficking.