Protein phosphatase 2A (PP2A) is a member of the intracellular serine/threonine phosphatases. Innate immune cell activation triggered by pathogen-associated molecular patterns is mediated by various protein kinases, and PP2A plays a counter-regulatory role by deactivating these kinases. In this study, we generated a conditional knockout of the alpha isoform of the catalytic subunit of PP2A (PP2ACalpha). After crossing with myeloid-specific cre-expressing mice, effective gene knockout was achieved in various myeloid cells. The myeloid-specific knockout mice (lyM-PP2Afl/fl) showed higher mortality in response to endotoxin challenge and bacterial infection. Upon LPS challenge, serum levels of TNF-alpha, KC, IL-6, and IL-10 were significantly increased in lyM-PP2Afl/fl mice, and increased phosphorylation was observed in MAPK pathways (p38, ERK, JNK) and the NF-kappaB pathway (IKKalpha/beta, NF-kappaB p65) in bone marrow-derived macrophages (BMDMs) from knockout mice. Heightened NF-kappaB activation was not associated with degradation of IkappaBalpha; instead, enhanced phosphorylation of the NF-kappaB p65 subunit and p38 phosphorylation-mediated TNF-alpha mRNA stabilization appear to contribute to the increased TNF-alpha expression. In addition, increased IL-10 expression appears to be due to PP2ACalpha-knockout-induced IKKalpha/beta hyperactivation. Microarray experiments indicated that the Toll/IL-1R domain-containing adaptor inducing IFN-beta/ TNFR-associated factor 3 pathway was highly upregulated in LPS-treated PP2ACalpha-knockout BMDMs, and knockout BMDMs had elevated IFN-alpha/beta production compared with control BMDMs. Serum IFN-beta levels from PP2ACalpha-knockout mice treated with LPS were also greater than those in controls. Thus, we demonstrate that PP2A plays an important role in regulating inflammation and survival in the setting of septic insult by targeting MyD88- and Toll/IL-1R domain-containing adaptor inducing IFN-beta-dependent pathways.