Human cytomegalovirus infection of monocytes stimulates a unique monocyte differentiation reprogramming resulting in polarization towards an M1 pro-inflammatory macrophage that simultaneously exhibits characteristics of an M2 anti-inflammatory macrophage. Our laboratory has previously shown that HCMV infection stimulates monocyte NF-kappaB and PI(3)K activities and now provides evidence that these cellular factors are essential for the HCMV-induced polarization of infected monocytes/macrophages. We find that the induction of NF-kappaB and PI(3)K activities following HCMV infection was required for the initiation of monocyte-to-macrophage differentiation. HCMV-infected monocytes treated with Bay11-7802 (an inhibitor of NF-kappaB activity) or LY294002 [an inhibitor of PI(3)K activity] prior to infection exhibited a small, round and monocyte-like undifferentiated morphology and the lack of CD68 upregulation (a macrophage differentiation marker). Detailed transcriptome analysis revealed 48%, 7% and 31% of HCMV-induced M1-associated genes were dependent on NF-kappaB, PI(3)K or both activities, respectively; while 100% of HCMV-induced M2-associated genes required both NF-kappaB and PI(3)K activities. Functionally, we demonstrated that NF-kappaB and PI(3)K activities were critical for the production of M1- and M2-associated cytokines/chemokines, in HCMV-induced differentiating monocytes. Supernatant from HCMV-infected monocytes pretreated with Bay11-7802 or LY294002 exhibited an 80% and 67% reduction in cell motility-inducing activity. Overall, these data show that HCMV usurps monocyte NF-kappaB and PI(3)K signal transduction pathways to induce the unique polarization of HCMV-infected monocytes needed for the earliest steps in the viral dissemination and persistence strategy.