Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative disorders that affect both humans and animals. They are characterised by the structural transformation of the cellular prion protein (PrPC) to the prion disease associated isoform (PrPSc). MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene or protein expression by targeting mRNAs and triggering either translational repression or mRNA degradation. Distinct miRNA signatures have been detected in exosomes released from various prion disease models. Decreased miR-146a levels were shown in exosomes released from prion infected cells and mice, as well as in the serum of Creutzfeldt-Jakob disease (CJD) patients. To investigate whether miR-146a plays a functional role in prion diseases, CRISPR/Cas9 mediated miR-146a knockdown in neuronal N2a cells, prion infection in N2a cells and in brain slices from miR-146a knockout mice were used to examine the effects of miR-146a gain/loss-of-function on prion proteins. miR-146a was shown to increase PrPC levels when overexpressed and downregulated in N2a cells. miR-146a overexpression decreased the amount of PrPSc in prion infected N2a cells, while prion infected brain slices from miR-146a knockout mice exhibited an increased PrPSc level compared to the wildtype mice. Prion protein encoding gene Prnp was also proved to be targeted by miR-146a in the 3’ untranslated region (3’ UTR). Overall, miR-146a plays a critical role in regulating prion proteins; exosome delivery of miR-146a may serve as a potential therapeutic in treating prion diseases.