Background and Aims: Inoculation of BALB/c mice with rhesus rotavirus (RRV) in the newborn period results in biliary epithelial cell (cholangiocyte) infection and the murine model of biliary atresia. Rotavirus infection of a cell requires attachment which is governed in part by cell surface expression of integrins such as 21. We hypothesized that cholangiocytes were susceptible to RRV infection because they express 21. Methods: RRV attachment and replication was measured in cell lines derived from cholangiocytes and hepatocytes. Flow cytometry was performed on these cell lines to determine if 21 was present. Cholangiocytes were blocked with natural ligands, a monoclonal antibody or siRNA against the 2 subunit and infected with RRV. The extra-hepatic biliary tract of newborn mice was screened for the expression of the 21 integrin. Newborn mice were pretreated with a monoclonal antibody against the 2 subunit and inoculated with RRV. Results: RRV attached and replicated significantly better in cholangiocytes than in hepatocytes. Cholangiocytes, but not hepatocytes, expressed 21 in vitro and in vivo. Blocking assays led to a significant reduction in attachment and yield of virus in RRV infected cholangiocytes. Pretreatment of newborn pups with an anti-2 monoclonal antibody reduced the ability of RRV to cause biliary atresia in mice Conclusion: Cell surface expression of the integrin 21 plays a role in the mechanism that confers cholangiocyte susceptibility to RRV infection.