Prion diseases are fatal transmissible neurodegenerative disorders that affect both human and animals. They are characterized by conformational conversion of the normal cellular prion protein (PrPc) into a disease associated pathological isoform (PrPSc). It has been shown that PrPSc can infect recipient cells when it is packaged into endosomal derived nanoparticles called exosomes. Exosomes are present in all biological fluids such as blood, urine, milk, and saliva. Exosome secretion is under the influence of the autophagic system, a basic cellular degradation machinery. Autophagy stimulation can inhibit exosomal release, whereas inhibition of autophagy seems to enhance exosomal release. In this paradigm, our work investigates the effect of autophagy modulation on exosomal release of prions and how this interplay impacts prion infection. We successfully isolated and characterized exosomes from a prion‐infected neuronal cell line (ScN2a) using differential ultracentrifugation technique. The isolated exosomes contained PrPSc as shown by immunoblot. Treatment of ScN2a cells with the neutral sphingomyelinase inhibitor GW4869 resulted in a block of the exosomal release and reduced PrPSc levels in exosomes. When we stimulated autophagy in ScN2a cells using rapamycin, a well‐known autophagy stimulator that inhibits the mammalian target of Rapamycin (mTOR) pathway, we observed a strong inhibition of exosomal release and decreased levels of PrPSc in exosomes compared to vehicle treated cells. To further assess the impact of autophagy on exosomal prion release, we knocked‐out Atg5 (a main player in the autophagic machinery) in N2a cells using the CRISPR‐Cas9 system. CRISPR‐Cas9 is the clustered, regularly interspaced, short palindromic repeats (CRISPR)‐associated protein (Cas) system which involves RNA–guided site specific DNA double strand cleavages. Non‐homologous end joining is used by cells for repair of double strand breaks and may result in functional gene knock‐out by introduction of insertions and deletions. Using this technology we generated various N2a cell clones with functional knockouts in different exons of the Atg5 gene, as verified by sequencing and immunoblot analysis. Upon stably infecting these Atg5 knock‐out cells with prions, we found a highly increased release of exosomes and exosome‐associated PrPSc compared to wild type cells. Taken together, our data shows that autophagy modulation can control lateral prion infection by interfering with exosomal release of PrPSc. Our present work correlates these findings to outcomes of prion infection in recipient cells and in mouse animal models. Overall, our study describes a novel interplay of basic cellular machinery which affects the live cycle of prions. This new understanding will result in novel targets for therapy against prion diseases and protein misfolding disorders.Support or Funding InformationThe Natural Sciences and Engineering Research Council of Canada (NSERC) Alberta Prion Research Institute (APRI) National Institute of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS) Alberta Innovates Health Solutions (AIHS)