Background Cell released microvesicles specifically, exosomes, play an important role in mediating immunologic escape, treatment resistance, and disease persistence of Hepatitis C virus (HCV) infection. Reports on the molecular compositions of exosomes released by cells under diverse conditions, especially during viral infections, suggest that their cargo contents are not randomly loaded. However, the precise molecular mechanisms directing the selective cargo sorting and loading inside infectious viral exosomes remains elusive. Aim To decipher the role of Reticulon 3 (RTN3) in the selective molecular cargo sorting and loading inside infectious viral exosomes during HCV infection. Methods We used Huh7 cells-JFH1 HCV infection and HCV Full-Length (FL) replicon systems. Additionally, we analyzed human liver and serum exosome samples from healthy and treatment na#239;ve HCV infected individuals. Our experiments made use of molecular biology and immunology techniques. Results HCV infection (JFH1-Huh7 or HCV-FL replicon cells) was associated with increased RTN3LS isoforms expression in cells and cell released exosomes. Accordingly, increased expression of RTN3LS was observed in liver and serum exosome samples of HCV infected individuals compared to healthy controls. RNA-ChIP analysis revealed that RTN3LS interacted with dsHCV RNA. Lentiviral CRISPR/Cas9 mediated knockdown (KD) of RTN3 and plasmid overexpression (OE) of wild type, C- and N-terminal deletion mutants of RTN3LS in HCV- infected Huh7 cells differentially impacted the cellular release of infectious viral exosomes. RTN3LS KD significantly decreased, while RTN3S OE significantly increased the number of Huh7 cell-released infectious exosomes. The C-terminal domain of RTN3 interacted with and modulated the loading of dsHCV RNA inside infectious exosomes. Antiviral treatment of HCV infected Huh7 cells reduced virus-induced RTN3LS expression and attenuated the release of infectious exosomes. Conclusion RTN3 constitutes a novel regulator and a potential therapeutic target that mediates the specific loading of infectious viral exosomes.