Infectious pancreatic necrosis virus (IPNV) is a viral disease that afflicts salmon aquaculture worldwide. Transmission of viral diseases may occur via the skin, gills or intestine but the main entry route for IPNV is not known. However, acute inflammation of the intestinal mucosa is an early symptom in infected fish which suggests transmission through an oral route. The main aim of this study is to determine if IPNV can translocate across the intestinal epithelium. It also aims to investigate how acute and chronic IPNV exposure as well as stress affects the physiological barrier functions of the intestinal epithelium. Possible translocation of IPNV across the intestinal mucosa as well as effects of virus on intestinal barrier function was measured in vitro, using the Ussing chamber technique. IPNV was translocated across both proximal and distal segments of the intestine with a higher rate in distal compared to proximal intestine. The translocated virions caused cytopathic effects on cultured Chinook salmon embryo cells (CHSE-214), proving viability and high virulence. Intestinal barrier function was disturbed as assessed by increased permeability for ¹⁴C-mannitol in the proximal region after exposure to IPNV in vitro. The same pattern was observed after prolonged exposure to IPNV in vivo. Further, the intestine appeared to respond with an increased mucus secretion, as indicated through increased TER after prolonged IPNV exposure. No additional effects of cortisol implantation on barrier function were observed. In conclusion, this study demonstrates that both proximal and distal intestine can be used by IPNV as a route of infection and that the intestinal barrier function is negatively affected by exposure to IPNV.