Myofibroblasts (MF) play an important role in intestinal wound healing. A compromised epithelial barrier exposes intestinal subepithelial MF to luminal bacterial products. However, responses of murine intestinal MF to bacterial adjuvants and potential roles of intestinal MF in innate immune responses are not well defined. Aims: To determine innate immune responses and intracellular signaling pathways of intestinal MF exposed to LPS, a prototypic TLR ligand. Expression of TLR-4 in primary murine intestinal MF cultures was confirmed by RT-PCR and western blotting. LPS-induced secretion of prostaglandin E₂ (PGE₂), interleukin (IL)-6, and keratinocyte-derived chemokine (KC) was measured by ELISA. Intracellular responses to LPS were assessed by western blotting for NFB p65, IB, Akt, p38 MAP kinase, and cyclooxygenase-2 (COX-2). LPS induced rapid phosphorylation of NFB p65, Akt, and p38 MAPK and degradation of IB. LPS induced expression of COX-2 and secretion of PGE2 (2.0±0.8 fold induction versus unstimulated cells), IL-6 (6.6±0.4 fold induction), and KC (12.5±0.4 fold induction). Inhibition of PI3-kinase, p38 MAPK or NFB pathways reduced LPS-induced PGE2, IL-6 and KC secretion. These studies show that primary murine intestinal MF respond to LPS, evidenced by activation of NFB, PI3-kinase and MAPK signaling pathways and secretion of proinflammatory molecules. Inhibition of these pathways attenuated LPS-dependent PGE2, IL-6 and KC production, indicating that LPS activates MF by multiple signaling pathways. These data support the hypothesis that MF are a component of the innate immune system and may exert paracrine effects on adjacent epithelial and immune cells by responding to luminal bacterial adjuvants.