Severe injury primes the innate-immune system for increased Toll-like receptor 4 (TLR4)-induced proinflammatory cytokine production by macrophages. In this study, we examined changes in TLR4 signaling pathways in splenic macrophages from burn-injured or sham mice to determine the molecular mechanism(s) responsible for the increased TLR4 responsiveness. Using flow cytometry and specific antibodies, we first looked for injury-induced changes in the expression levels of several TLR-associated signaling molecules. We found similar levels of myeloid differentiation primary-response protein 88 (MyD88) and interleukin-1 receptor-associated kinase-M (IRAK-M) and somewhat lower levels of total p38, extracellular signal-regulated kinase (ERK), and stress-activated protein kinase (SAPK)/c-jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) in burn compared with sham macrophages. However, with the use of antibodies specific for the phosphorylated (activated) forms of the three MAPKs, we found that macrophages from burn mice showed a twofold increase in purified lipopolysaccharide (LPS)-stimulated p38 activation as compared with cells from sham mice on days 1 and 7 post-injury, whereas ERK and SAPK/JNK activation was increased by burn injury only on day 1. Using the specific p38 inhibitor (SB203580), we confirmed that the increase in tumor necrosis factor alpha production by LPS-stimulated burn macrophages requires p38 activation. Although we demonstrated that injury increases macrophage TLR4 mRNA expression and intracellular expression of TLR4-myeloid differentiation protein-2 (MD-2) protein, macrophage cell-surface expression of TLR4-MD-2 was not changed by burn injury. Our results suggest that the injury-induced increase in TLR4 reactivity is mediated, at least in part, by enhanced activation of the p38 signaling pathway.