We have established that HCMV acts as a specific ligand engaging and activating cellular integrins on monocytes. As a result, integrin signaling via Src activation leads to the functional activation of paxillin required for efficient viral entry and for the biological changes in monocytes needed for viral dissemination. These biological/molecular changes allow HCMV to use monocytes as "vehicles" for systemic spread and the establishment of lifelong persistence. However, it remains unresolved how HCMV specifically induces this observed monocyte activation. It was previously demonstrated that the HCMV gH/gL/UL128-131 glycoprotein complex facilitates viral entry into biologically relevant cell types. Nevertheless, the mechanism by which the gH/gL/UL128-131 complex promotes this process is unknown. We now show that only HCMV virions possessing the gH/gL/UL128-131 complex are capable of activating integrin/Src/paxillin-signaling in monocytes. In fibroblasts, this signaling is reversed, such that virus lacking the gH/gL/UL128-131 complex is the only virus able to induce the paxillin activation cascade. The presence of the gH/gL/UL128-131 complex also may have an inhibitory effect on integrin-mediated signaling pathway in fibroblasts. Furthermore, we demonstrate that the presence of the gH/gL/UL128-131 complex on the viral envelope, through its activation of the integrin/Src/paxillin pathway, is necessary for efficient HCMV internalization into monocytes and that appropriate actin and dynamin regulation is critical for this entry process. Importantly, productive infection in monocyte-derived macrophages was seen only in cells exposed to HCMV expressing the gH/gL/UL128-131 complex. From our data, the HCMV gH/gL/U128-131 complex emerges as the specific ligand driving the activation of the receptor-mediated signaling required for the regulation of the actin cytoskeleton and, consequently, for efficient and productive internalization of HCMV into monocytes. To our knowledge, our studies demonstrate a possible molecular mechanism for why the gH/gL/UL128-131 complex dictates HCMV tropism and why the complex is lost as clinical isolates are passaged in the laboratory.