Phosphatidylinositol-3-phosphate kinase (PI3K) has been reported to exhibit anti-inflammatory roles as a negative modulator of the NF-kappaB pathway (MyD88- and Mal-dependent) triggered upon Toll-like receptor (TLR)4 activation by lipopolysaccharide (LPS). Here, we investigated the role of PI3K on the TLR4-dependent, MyD88-independent signaling cascade which is activated in macrophages infected by Vesicular Stomatitis Virus (VSV) and leads to interferon production, thus conferring antiviral protection. We show that VSV induces TLR4 (and CD14)-dependent Akt phosphorylation. We observed hypersusceptibility to viral infections after pharmacological inactivation of the PI3K pathway in macrophages, which indicates that normal PI3K functions are critical for type I interferon synthesis and viral resistance. Conversely, we noticed increased resistance in macrophages isolated from genetically modified mice in which the PI3K pathway is constitutively active. Our data, which demonstrate that PI3K-Akt axis is an important component of the TLR4-dependent antiviral mechanism, also indicate that pharmacological modulation of this pathway to regulate the inflammatory response could promote viral susceptibility.