The control of viral infections in the brain involves the activation of microglial cells, the macrophages of the brain that are constantly surveying the central nervous system, and the production of amyloid-beta (Aβ) as an anti-microbial molecule. Recent findings suggest a possible implication of HHV-6A in AD. We evaluated the effect of HHV-6A infection on microglial cell expression Aβ and the activation status, determined by TREM2, ApoE, cytokines, and tau expression. We have infected microglial cells (HMC3, ATCC®CRL-3304), in monolayer and human peripheral blood monocyte-derived microglia (PBM-microglia) spheroid 3D model, with HHV-6A (strain U1102) cell-free virus inocula with 100 genome equivalents per 1 cell. We collected the cells 1, 3, 7, and 14 days post-infection (d.p.i.) and analyzed them for viral DNA and RNA, ApoE, Aβ (1-40, 1-42), tau, and phospho-tau (Threonine 181) by real-time immunofluorescence and cytokines by immunoenzymatic assay. We observed a productive infection by HHV-6A. The expression of Aβ 1-42 increased from 3 d.p.i., while no significant induction was observed for Aβ 1-40. The HHV-6A infection induced the activation (TREM2, IL-1beta, ApoE) and migration of microglial cells. The secretion of tau started from 7 d.p.i., with an increasing percentage of the phosphorylated form. In conclusion, microglial cells are permissive to HHV-6A infection that induces the expression of Aβ and an activation status. Meanwhile, we hypothesize a paracrine effect of HHV-6A infection that activates and induces microglia migration to the site of infection.