IFN--driven and CD8+ T cells-dependent inflammatory injury to extrahepatic biliary epithelium (EHBE) is likely to be involved in the development of biliary atresia (BA). We previously showed that viral protein NSP4 is the pathogenic immunogen which causes biliary injury in BA. In this study, NSP4 or four synthetic NSP4 (NSP4_157-170, NSP4_144-152, NSP4_93-110, NSP_424-32) identified by computer analysis as candidate CD8+ T cells epitopes were injected into neonatal mice. The pathogenic NSP4 epitopes were confirmed by studying extrahepatic bile duct injury, IFN- release and CD8+ T cell response against EHBE. The results revealed, at 7 days post-injection (dpi), inoculation of GST-NSP4 caused EHBE injury and BA in neonatal mice. At 7 or 14 dpi, inoculation of GST-NSP4, NSP4_144-152 or NSP4_157-170 increased IFN- release by CD8+ T cells, elevated the population of hepatic memory CD8+ T cells, and augmented cytotoxicity of CD8+ T cells to rhesus rotavirus (RRV)-infected or naïve EHBE cells. Furthermore, depletion of CD8+ T cells in mice abrogated the elevation of GST-NSP4-induced serum IFN-. Lastly, parenteral immunization of mouse dams with GST-NSP4, NSP4_144-152 or NSP4_157-170 decreased the incidence of RRV-induced BA in their offspring. Overall, this study reports the CD8+ T cell response against EHBE is activated by epitopes within rotavirus NSP4 in experimental BA. Neonatal passive immunization by maternal vaccination against NSP4_144-152 or NSP4_157-170 is effective in protecting neonates from developing RRV- related BA.