Phosphatidylinositol-3-kinase (PI3K)/Akt is an important cellular pathway that has been shown to participate in various replication steps of multiple viruses. In the present study, we compared the phosphorylation status of Akt during infection of MARC-145 cells and porcine alveolar macrophages (PAMs) with highly pathogenic PRRSV (HP-PRRSV) strain HuN4. We observed that biphasic activation of Akt was induced in at both the early stage (5, 15 and 30 min postinfection) and the late stage (12 and 24 h postinfection) of HP-PRRSV infection of MARC-145 cells, while an early-phase activation of Akt was found exclusively in virus-infected PAMs in vitro. Analysis with the PI3K-specific inhibitor LY294002 confirmed that PI3K acted as the upstream activator for the virus-induced activation of Akt. UV-irradiation-inactivated virus still induced the early event in PAMs but not in MARC-145 cells, suggesting that different mechanisms are employed for the early-stage induction of phosphorylated Akt within different cell cultures. We further demonstrated that FoxO1 and Bad, which serve as downstream targets of Akt, were phosphorylated in virus-infected MARC-145 cells. Moreover, the suppression of phosphorylated Akt with LY294002 significantly inhibited the virus-induced cytopathic effect (CPE) on MARC-145 cells, but it had a negligible effect on virus propagation. Collectively, our data provide new evidence of a novel role for the PI3K/Akt pathway in PRRSV infection of MARC-145 cells.