<p>Cytomegalovirus is the leading viral cause of congenital infection with neurological sequelae. Effective medical treatments are limited due to an inadequate understanding of the underlying pathogenesis. Here, we applied single-cell transcriptomics to analyze neonatal mouse brains with congenital cytomegalovirus infection (cCMV). We profiled cCMV in 22 cell types and identified neural progenitor cells (NPCs) and monocyte-derived macrophages (MDMs) as the most commonly infected cells. Infected NPCs exhibited dysregulated neurodevelopment-associated signaling pathways, correlating with viral transcript levels that indicate viral replication levels. Genes associated with phagocytosis and antigen presentation were downregulated exclusively in infected MDMs but remained largely unaffected in microglia and barrier-associated macrophages regardless of infection status. Analysis of intrinsic and induced interferon-stimulated gene expression revealed great heterogeneity across cell types but no direct correlation with cCMV susceptibility. Furthermore, our findings indicate that interferon type II is crucial for the control of cCMV and consequent cortical damage and calcification in the neonatal brain. This study advances our understanding of cCMV tropism and the molecular details of cCMV-induced neurodevelopmental impairment, cerebral immune response, and brain pathology.</p>