Background: biliary atresia (BA) is a devastating neonatal cholangiopathy that leads to cholestasis and progressive hepatic failure. Our poor understanding of the onset and progression of BA results in compromised transplant-free survival. Osteopontin (OPN, encoded by SPP1), is highly expressed in biliary epithelial cells (BECs) and is involved in chronic liver disease, however whether OPN participates in BA is unknown. Here we investigated the role of OPN in the onset and progression of BA. Methods: SPP1 gene expression in BA patients was analyzed using publicly available datasets of scRNA-seq, RNA-seq, and microarrays. OPN protein expression in BA livers was analyzed by immunohistochemistry. OPN concentration in serum was measured by ELISA. To induce BA in mice, we inoculated i.p. rhesus rotavirus (RRV) to BALB/c pups as well as pups with Spp1 depletion in the liver within 24 hours of birth. Symptoms of BA were monitored after RRV inoculation and mice were sacrificed on day 11. Growth rate was evaluated, activity of transaminases and levels of total bilirubin were measured in serum, and histopathological changes were assessed. Results: in humans, SPP1 is predominantly expressed in BECs in healthy individuals and is highly increased in cirrhosis. Notably, SPP1 gene expression is significantly higher in livers from BA patients compared to non-BA cholestatic liver disease or control pediatric liver donors (GSE122340, GSE46960). SPP1 gene expression highly correlates with KRT7, KRT19 and MMP7, all markers of BECs. BA patients with fibrosis exhibit higher SPP1 expression than those with inflammation only (GSE15235). Liver tissue from BA patients shows prominent expression of OPN. The OPN concentration in serum is significantly higher in BA patients compared with patients with other cholestatic liver diseases. BALB/c mice with BA show delayed development, jaundice and acholic stools. Moreover, they have increased transaminases and higher levels of total bilirubin. H&E staining reveals massive ductular reaction and hepatic inflammation. The concentration of OPN in serum and urine and the hepatic expression of OPN are significantly increased in mice with BA compared to controls. Mice lacking Spp1 in the liver exhibit significant body weight reduction as well as more inflammatory response in liver tissue 11 days after RRV injection compared with control littermates. Conclusions: hepatic SPP1 gene expression and serum OPN concentration differentiate BA from patients with other cholestatic liver disease. OPN is highly expressed in serum, urine, and liver tissue in a mouse model of BA. Depletion of Spp1 from liver results in significant grow retardation and an inflammatory response after RRV injection.