<p>siRNA therapeutics have achieved increased clinical success in the past decade. To achieve a therapeutic effect, siRNAs must enter the cytoplasm of the target cells. Thus, their function typically relies on proper endocytosis and trafficking. Endoplasmic reticulum (ER) stress is associated with diseases that are being studied for treatment with siRNAs (cancer, amyloids, mutations, etc.) and common comorbidities (obesity, smoking, hypertension, etc.). ER stress results in activation of the Unfolded Protein Response (UPR), which initiates changes in cellular function that include disruption to endosomal vesicle trafficking and processing. We used the N-glycosylation inhibitor, tunicamycin, to induce ER stress in HeLa cells during transfection with siRNAs targeting EGFP. Our results showed that ER stress resulted in an increased accumulation of siRNA but a reduction in silencing of the siRNA target (EGFP). Further, we used 3-methyladenine (autophagy inhibitor) and bafilomycin A1 (inhibits endosome maturation) to show that autophagy plays a role in increasing siRNA accumulation. Finally, we showed that the additional accumulated siRNA in ER-stressed cells remained functional, was retained in cells for longer, and prolonged silencing. Our results will inform the design of siRNA delivery vehicles and dosing schedules, by ensuring that disease complications, specifically ER stress/UPR activation, are considered.</p> Graphical abstract <p></p>