AbstractClusterin (CLU) is one of the most significant genetic risk factors for late onset Alzheimer’s disease (AD). However, the mechanisms by which CLU contributes to AD development and pathogenesis remain unclear. Studies have demonstrated that the trafficking and localisation of glycosylated CLU proteins is altered by CLU-AD mutations and amyloid-β (Aβ), which may contribute to AD pathogenesis. However, the roles of non-glycosylated and glycosylated CLU proteins in mediating Aβ toxicity have not been studied in human neurons. iPSCs with altered CLU trafficking were generated following the removal of CLU exon 2 by CRISPR/Cas9 gene editing. Neurons were generated from control (CTR) and exon 2 −/− edited iPSCs and were incubated with aggregated Aβ peptides. Aβ induced changes in cell death and neurite length were quantified to determine if altered CLU protein trafficking influenced neuronal sensitivity to Aβ. Finally, RNA-Seq analysis was performed to identify key transcriptomic differences between CLU exon 2 −/− and CTR neurons. The removal of CLU exon 2, and the endoplasmic reticulum (ER)-signal peptide located within, abolished the presence of glycosylated CLU and increased the abundance of intracellular, non-glycosylated CLU. While non-glycosylated CLU levels were unaltered by Aβ_25–35 treatment, the trafficking of glycosylated CLU was altered in control but not exon 2 −/− neurons. The latter also displayed partial protection against Aβ-induced cell death and neurite retraction. Transcriptome analysis identified downregulation of multiple extracellular matrix (ECM) related genes in exon 2 −/− neurons, potentially contributing to their reduced sensitivity to Aβ toxicity. This study identifies a crucial role of glycosylated CLU in facilitating Aβ toxicity in human neurons. The loss of these proteins reduced both, cell death and neurite damage, two key consequences of Aβ toxicity identified in the AD brain. Strikingly, transcriptomic differences between exon 2 −/− and control neurons were small, but a significant and consistent downregulation of ECM genes and pathways was identified in exon 2 −/− neurons. This may contribute to the reduced sensitivity of these neurons to Aβ, providing new mechanistic insights into Aβ pathologies and therapeutic targets for AD.