Problem: Down‐modulation of human leukocyte antigen (HLA)‐G1 cell surface expression by human cytomegalovirus (HCMV) has only been studied in cellular models expressing independent unique short (US) recombinant proteins, but not in the context of viral infection. To explore the level of HLA‐G1 cell surface expression after HCMV infection and to investigate the influence of US viral proteins, we infected HLA‐G1 expressing cells by HCMV laboratory strains.Method of study: Human U373‐MG astrocytoma cells were transfected with HLA‐G1 cDNA. Following HCMV infection, HLA‐G1 cell surface expression of these transfectants was evaluated by flow cytometry and confocal microscopy, using an HLA‐G specific monoclonal antibody, and compared with that of uninfected cells. US‐deleted viruses were then used to evaluate the implication of US proteins.Results: Using flow cytometry, it was found that HCMV infection of U373‐G1 cells decreased HLA‐G1 cell surface expression. Similar results were obtained with two different HCMV strains, namely Towne and AD169. Two color confocal microscopy staining further confirmed such HLA‐G down‐modulation in HCMV‐infected cells stained for immediate early (IE1/2) nuclear proteins expression. Infection of U373‐G1 cells with US‐deleted HCMV strain had no effect on the level of cell surface HLA‐G1 expression, thus demonstrating the US dependency of the HCMV‐mediated down‐regulation of HLA‐G1.Conclusion: HCMV infection down‐modulates HLA‐G1 expression at the cell surface. This is likely to have functional consequences in case of HCMV uterine infection during pregnancy.