Severe injury deranges immune function and increases the risk of sepsis and multiple organ failure. Kupffer cells play a major role in mediating posttraumatic immune responses, in part via different Toll-like receptors (TLR). Although mitogen-activated protein kinases (MAPK) are key elements in the TLR signaling pathway, it remains unclear whether the activation of different MAPK are TLR specific. Male C3H/HeN mice underwent midline laparotomy (i.e., soft tissue injury), hemorrhagic shock (MAP ∼35 mm Hg for 90 min), and resuscitation. Kupffer cells were isolated 2 h thereafter, lysed and immunoblotted with antibodies to p38, ERK1/2, or JNK proteins. In addition, cells were preincubated with specific inhibitors of p38, ERK1/2, or JNK MAPK followed by stimulation with the TLR2 agonist, zymosan; the TLR4 agonist, LPS; or the TLR9 agonist, CpG DNA. Cytokine (TNF-α, interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and KC) production was determined by cytometric bead array after 24 h in culture. MAPK activity as well as TNF-α, MCP-1, and KC production by Kupffer cells were significantly increased following trauma-hemorrhage. TLR4 activation by LPS stimulation increased the levels of all measured cytokines. CpG-stimulated TLR9 signaling increased TNF-α and IL-6 levels; however, it had no effect on chemokine production. Selective MAPK inhibition demonstrated that chemokine production was mediated via p38 and JNK MAPK activation in TLR2, -4, and -9 signaling. In contrast, TNF-α and IL-6 production was differentially regulated by MAPK depending on the TLR pathway stimulated. Thus, Kupffer cell TLR signaling employs different MAPK pathways in eliciting cytokine and chemokine responses following trauma-hemorrhage. J. Cell. Physiol. 210: 667–675, 2007. © 2006 Wiley-Liss, Inc.