After infection, human CMV (HCMV) establishes a latent and persistent infection in immature myeloid progenitors and peripheral blood monocytes. Completion of the HCMV life cycle is possible upon maturation of monocytes to tissue macrophages and under permissive circumstances, e.g., immunosuppression. We investigated the hypothesis that HLA-G molecules could be induced during HCMV reactivation in activated macrophages to favor virus dissemination. In this study, we provide evidence that HLA-G Ags are produced during viral reactivation in macrophages generated after allogeneic stimulation of HCMV latently infected monocytes. While HLA-G surface expression is up-regulated, classical MHC-I molecules are partially down-regulated by HCMV. In vivo, bronchoalveolar macrophages collected from patients suffering from acute HCMV pneumonitis also express HLA-G molecules. The direct correlation between HLA-G Ag induction and HCMV infection was confirmed in U-373 MG astrocytoma cells. Soluble HLA-G expression is stimulated upon HCMV infection, and this modulation depends on the cooperative action of the two immediate-early-1 pp72 and immediate-early-2 pp86 products. Because HLA-G transcription is active in macrophages and U-373 MG astrocytoma cells, it is likely that the modulation of HLA-G protein expression during HCMV replication occurs at a post-transcriptional level. Our data suggest that induction of HLA-G molecules could be an additional mechanism that helps HCMV to subvert host defenses.