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Macrophages (MΦ) play a role in neonatal etiologies of obstructive cholestasis, however, the role for precise MΦ subsets remains poorly defined. We developed a neonatal murine model of bile duct ligation (BDL) to characterize etiology-specific differences in neonatal cholestatic MΦ polarization. Neonatal BDL surgery was performed on female BALB/c mice at 10 days of life (DOL) with sham laparotomy as controls. Comparison was made to the Rhesus Rotavirus (RRV)-induced murine model of biliary atresia (BA). Evaluation of changes at day 7 after surgery (BDL and sham groups) and murine BA (DOL14) included laboratory data, histology (H&E, anti-CD45 and anti-CK19 staining), flow cytometry of MΦ subsets by MHCII and Ly6c expression, and single cell RNA-sequencing (scRNA-seq) analysis. Neonatal BDL achieved a 90% survival rate; mice had elevated bile acids, bilirubin, and alanine aminotransferase (ALT) versus controls (p < 0.05 for all). Histology demonstrated hepatocellular injury, CD45+ portal infiltrate, and CK19+ bile duct proliferation in neonatal BDL. Comparison to murine BA showed increased ALT in neonatal BDL despite no difference in histology Ishak score. Neonatal BDL had significantly lower MHCII-Ly6c+ MΦ versus murine BA, however, scRNA-seq identified greater etiology-specific MΦ heterogeneity with increased endocytosis in neonatal BDL MΦ versus cellular killing in murine BA MΦ. We generated an innovative murine model of neonatal obstructive cholestasis with low mortality. This model enabled comparison to murine BA to define etiology-specific cholestatic MΦ function. Further comparisons to human data may enable development of immune modulatory therapies to improve patient outcomes. [ABSTRACT FROM AUTHOR]